Physiologically Relevant Cell Culture handbook by AMSBIO

Physiologically relevant cell culture Since the middle of the 20th century, cell biology has relied heavily on tissue culture techniques and animal models. Tissue culture techniques frequently involve culture of long-term cell lines on plastic surface. These techniques, sometimes called two-dimensional or 2D, culture employ relatively high levels of oxygen and serum. While these techniques are successful in supporting many cell lines, there is increasing concern that they provide an inadequate model for in vivo physiological contexts. Similarly, animal models such as xenografts, while they are an in vivo experimental system, are often not performed in the right disease context and therefore lack some of the natural microenvironment of the cells studied. Traditionally, natural hydrogels (basement membrane extract or individual extracellular proteins) have been used to create 2D, and more recently three-dimensional (3D) environments in vitro – this subject is covered in Natural Extracellular Matrix Proteins section. Nowadays, basement membrane extract variants also exist to enhance the physiological context of specific culture systems, including organoids, stem cells and xenografts. The use of artificial scaffolds for 3D culture (Artificial Scaffolds & Matrices section) such as Alvetex® is particularly prevalent in regenerative medicine. These are known to support generations of various tissue types, such as liver, skin and neurons. This is complemented by the MAPTrix™ range of biomimetics, allowing tailoring of tissue culture conditions by addition of signals from specific extracellular proteins and/or growth factors. The Scaffold Free Solutions section covers the emergence of spheroid-based assays as quantitative approaches to physiologically relevant cell models. These assays generate a microenvironment in a tight cluster of cells which can be detected by microscopy and performed quickly on high-throughput scale. Two-dimensional cell behaviour assays provide unrivalled throughput as drug development platforms, but are not strongly predictive of drug activity in the clinical setting. It has become clear that providing a physiological context in these assays is a way to improve on the high attrition rates frequently observed in clinical trials. This can be achieved by introduction of three-dimensional matrices, bio-mimetic arrays and other approaches covered in Cell Behaviour Assays section.

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PHYSIOLOGICALLY RELEVANT CELL CULTURE HANDBOOK TA B L E O F C O N T E N T S

Natural Extracellular Matrix Proteins

Cultrex Basement Membrane Extract (BME) 5 Cultrex® BME 1, PathClear®

Technology Highlight Organoid and Xenograft Matrices

Cultrex® Human Basement Membrane Extract (BME)

Technology Highlight: Cultrex® Directed In Vivo Angiogenesis Assay (DIVAA)

3D and Stem Cell Qualified Extracellular Membrane Proteins 9 Cultrex® 3D Culture Matrix Collagen I

Cultrex® 3D Culture Matrix Laminin I

Cultrex® BME and Extracellular Matrix Proteins – 3D and Stem Cell Qualified

Individual Extracellular Matrix Proteins and Solutions 11 Cultrex® Mouse Laminin I, PathClear®

Cultrex® Poly-L-Lysine

Cultrex® Poly-D-Lysine

Cultrex® Collagen I

Cultrex® Mouse Collagen IV

Cultrex® Human Fibronectin, PathClear®

Cultrex® Human Vitronectin, PathClear®

Artificial Scaffolds & Matrices

Alvetex® SCAFFOLD

Co-culture & Perfusion Solutions using Alvetex®

Alvetex® STRATA

Alginate

MAPTrix™ - Recombinant Animal Free Extracellular Matrix

Scaffold Free Solutions

Lipidure®- COAT low-attachment solutions

Spheroid-based Assays

Proliferation/Viability Assays

Cell Behaviour Assays

MAPTrixTM Adhesion Arrays

CultreCoat® Cell Adhesion Assays

Cultrex® Cell Invasion Assays

CultreCoat® Cell Invasion Optimization System

Oris™ Cell Migration Assays

Other Physiologically Relevant Cell Assays

Cultrex® 3-D Proliferation Assays

Cultrex® In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit

CultreCoat® Vascular Permeability Assays

Cultrex® 3-D Culture Cell Harvesting Kit

Cultrex® Cell Staining Kit

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Natural Extracellular Matrix Proteins

The extracellular matrix (ECM) is the non-cellular environment of the cell that directs multicellular organization and provides structural support for tissues. The ECM provides anchorage for cells and connects directly to the cell cytoskeleton through trans-membrane receptors. These physiological interactions control vital cell functions, such as proliferation, differentiation, migration, polarity, and survival. These processes are regulated through modulation of the cell’s epigenetic program and signal transduction cascades. Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma. Basement membranes are degraded and regenerated during development and wound repair. They not only support cells and cell layers, but they also play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Basement membranes provide major barriers to invasion by metastatic tumour cells ECM proteins have revolutionised in vitro and in vivo cell models by providing optimal environmental conditions to promote physiologically relevant cellular structure and function. These proteins have been used to: • • • • • •

Develop several organotypic models using 3D culture Provide barriers Evaluate metastatic potential Improve cellular implantation and evaluate angiogenesis in vivo Maintain stem cells in an undifferentiated state Induce stem cell differentiation

The PathClear® designation for EHS derived material means that in addition to standard sterility, endotoxin and MAP testing, the basement membrane extract is tested by PCR and is clear of 31 pathogens and viruses, including lactate dehydrogenase elevating virus (LDEV), which replicates in macrophages. Each lot is rigorously qualified in biological perfomances assays. PathClear® sterility testing for human materials includes: No bacterial or fungal growth detected after incubation at 370C for 14 days following USP XXIV Chapter 71 sterility test; no mycoplasma contamination detected by PCR; and endotoxin concentrations ≤ 20 EU/ml by LAL assay. All materials tested negative for eight human pathogenic viruses including Hepatitis A, B and C, HIV 1 and 2, Hantaan, Seoul and Sin Nombre by PCR.

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Cultrex Basement Membrane Extract READ OUTS

MAIN BENEFITS • • • • •

BME 1 is the gold standard product for 3D cell culture in vitro 3D and stem cell specific BME formulations are batch-qualified for these applications BME 2 is specially formulated to support organoid culture BME 3 is designed to enhance xenograft growth in vivo Ability to work with matrix consistent with the origin of human cell lines

Cat. No

• • • • • •

Viability/cytotoxicity Invasion and motility Cell signalling and communication Tissue regeneration Imaging: immunofluorescence, confocal and electron microscopy Retrieval of DNA, and RNA for gene analysis

Description

Pack

MOUSE BME BME 1 3432-001-01

Cultrex® BME 1 (Basement Membrane Extract, PathClear®)

1 ml

3432-005-01

5 ml

3432-010-01

2x 5ml

3433-001-01 3433-005-01

Cultrex® BME 1, RGF (Reduced Growth Factor, Basement Membrane Extract, PathClear®)

3433-010-01

1 ml 5 ml 2x 5ml

3D and Stem Cell Qualified Formats of BME 1 3445-001-01 3445-005-01

BME 1 3D Culture Matrix™ RGF (3D qualified, Basement Membrane Extract, Reduced Growth Factor, PathClear®)

3445-010-01 3434-001-02 3434-005-02

1 ml 5 ml 2x 5ml

Cultrex® Stem Cell Qualified BME 1, RGF (Reduced Growth Factor, Basement Membrane Extract, PathClear®)

1 ml 5 ml

BME 2 3532-001-02

Cultrex® BME 2 (Basement Membrane Extract, Type 2, PathClear®)

1 ml

3532-005-02

5 ml

3532-010-02

2x 5ml

3533-001-02 3533-005-02

Cultrex® BME 2 Organoid Matrix, RGF (Reduced Growth Factor, Basement Membrane Extract, Type 2, PathClear®)

3533-010-02

1 ml 5 ml 2x 5ml

BME 3 3632-001-02 3632-005-02

Cultrex® BME 3 Xenograft Matrix (Basement Membrane Extract, Type 1 ml 3, PathClear®) 5 ml

3632-010-02

2x 5ml HUMAN BME

3415-001-03

Cultrex® Stem Cell Qualified Human BME, PathClear® 5 www.amsbio.com

1 mg/ 1ml

Cultrex® BME 1, PathClear®

Cultrex® Basement Membrane Extract (BME 1) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumour. The extract gels at 37°C to form a reconstituted basement membrane. The major components of BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan. BME can be used in a multiple applications, under a variety of cell culture conditions, for maintaining growth or promoting differentiation of primary endothelial, epithelial, smooth muscle and stem cells. BME can also be utilized in cell attachment, neurite outgrowth, angiogenesis, in vitro cell invasion and in vivo tumourigenicity assays. BME typically ranges in protein concentrations from 14 to 16 mg/ml. PathClear® BME is available in normal and reduced growth factor form and is ideal for in vivo murine research work and other work requiring BME free from viruses, bacteria and mycoplasma.

Fig.1 SVEC4-10 endothelial cells stained using Cell Staining Kit ( cat. no #3437-100-K)

Technology Highlight Organoid and xenograft matrices Organoid matrix BME 2 Reduced Growth Factor

Xenograft matrix BME 3

• •

Higher tensile strength than original matrigel™ or BME Formulated for Organoid growth (3D stem cell culture)

Physiologically aligned with in vivo tumor environment Recommended for PDX and other xenograft

Cultrex BME Selection: Comparison of BME 1, 2 and 3 Name

Buffer

Tensile Strength

Concentration

Applications

BME 1

DMEM

MEDIUM

14-17 mg/ml

Xenograft/tumorgraft 2D cell culture 3D spheroids/Organoid Stem cells

BME 2

DMEM

HIGH

14-17 mg/ml

Reduced growth factor Format optimized for Organoid Growth

BME 3

RPMI1640

HIGH

14-17 mg/ml

Optimised for Xenograft/tumorgraft

*** BME 1, BME 2 and BME 3 are all derived from mouse EHS tumour ***

Fig.2 NCaP ATCC prostate adenocarcinoma cells (ATCC® CRL-1740™) were cultured in growth-factor reduced BME2 (4 mg/ml, prepared in phenol red-free RPMI-1640 medium) for 18 days. Culture medium was phenol red-free RPMI-1640 supplemented with 10% FBS, 1% penicillium/streptomycin and 2 mM L-glutamin. Living cells were stained 1h at +37°C with 1 µM Calcein AM and dead cells with 3 µM Ethidium homodimer-1. The 3D stack images were acquired using Zeiss Axiovert 200 M with spinning disc confocal unit Yokogawa CSU22 and a Zeiss Plan-Neofluar 5x objective. Z-stacks were acquired with a step-size of 40 µm. Maximum intensity projections were created by SlideBook 4.2.0.7 and background was subtracted with NIH ImageJ. Image courtesy of Matthias Nees and Ville Härmä (VTT Turku, Finland).

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Cultrex® Human Basement Membrane Extract (BME) Cultrex® Human Basement Membrane Extract (BME) is a soluble form of basement membrane purified from human placenta. BME can be used for promotion and maintenance of an undifferentiated phenotype, including stem cells, primary epithelial cells, endothelial cells and smooth muscle cells. It has been employed in cell attachment assays, neurite outgrowth assays, and tumour cell invasion assays. Immunostaining of H9 hESCs cultured on Cultrex® StemCell Qualified Human BME

Fig. 3 H9 human embryonic stem cells after three passages on Cultrex® Stem Cell Qualified Humam BME, PathClear® maintain expression of pluripotency markers Oct-4 (A) and Nanog (B). Nuclear staining by DAPI shown on panel © and merged image of Oct-4, Nanog and DAPI shown on panel (D). Images courtesy of the Yanik lab, MIT www.rle.mit.edu/bbng

Cat. no 3415-001-03

Description Cultrex® Stem Cell Qualified Human Basement Membrane Extract, PathClear®

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Pack 1 ml

Technology Highlight Cultrex® Directed in vivo Angiogenesis Assay (DIVAATM) DIVAATM is the first in vivo system for the study of angiogenesis that provides quantitative and reproducible results. During the course of the assay, implant grade silicone cylinders closed at one end, called angioreactors, are filled with 20 μl of PathClear® basement membrane extract (BME) premixed with or without angiogenic-modulating factors. These angioreactors are then implanted subcutaneously in the dorsal flank of nude mice. Accompanied with the onset of angiogenesis, vascular endothelial cells proceed to grow into the BME and form vessels in the angioreactor. As early as nine days post-implantation, there are enough cells to determine an effective dose response to angiogenic modulating factors. The sleek design of the angioreactor provides a standardized platform for reproducible and quantifiable in vivo angiogenesis assays. Compared to the plug assay, the angioreactor prevents assay errors due to absorption of the basement membrane extract by the mouse. In addition, the angioreactor uses only a fraction of the materials conserving both BME and test compounds used, and up to four angioreactors may be implanted in each mouse, allowing for greater statistical power. DIVAATM has been used in a variety of investigations. Cat. no 3450-048-DA

Description Cultrex® DIVAA

Pack

Angioreactor and BME

48 Wells

DIVAATM is available in three formats: DIVAATM Starter Kit # 3450-048-SK

The DIVAATM Starter Kit was designed to introduce the technology and give the user practical experience assessing angiogenesis. It contains 48 Angioreactors, enough growth factor to induce angiogenesis all 48 angioreactors, and an Angiorack™ designed to hold the angioreactors during the course of assay setup.

DIVAATM Activation Kit # 3450-048-K

The DIVAATM Activation Kit was designed for assessing angiogenesis activation. It contains 48 angioreactors and enough growth factor for eight positive controls.

DIVAATM Inhibition Assay # 3450-048-IK

The DIVAATM Inhibition Kit was designed for assessing angiogenesis inhibition. It contains 48 angioreactors and enough growth factor to induce angiogenesis in all 48 angioreactors.

READ OUTS • • •

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Vessel density Vessel length Vessel sprouting

3D and Stem Cell Qualified Extracellular Membrane Proteins Cultrex® 3D Culture Matrix™ BME and proteins are prequalified for the ability to support the culture of cells in three dimensions. 3D culture is an innovative approach to modelling the morphological effects of early oncogenesis on glandular epithelial cells. When healthy, these cells exhibit a structured, polarized morphology that is critical for tissue architecture and function. During carcinoma development, cell cycle controls associated with cellular development, proliferation and death are lost, and as a result, acinar structure formation is disrupted. In effect, the morphology of these structures can be used as a measure to study factors in early carcinoma development. MAIN BENEFITS • •

Basement Extract Matrices and derived proteins batch-qualified for 3D cell culture for 3D cell culture Matrices and proteins also suitable for stem cell work Fig.4 Non transgenic primary mammary cells grown in Cultrex® Matrix develop into a polarized acinus. Confocal microscopy ( 5µm projection) demonstrates epithelial polarity: DAPI stain, blue:GM130, red Golgi protein, apical marke; panel A) Z01, green (tight junctions, apical; panel B); Integrin a6, magenta (baso-lateral; panel C), overlay shown in panel D. Images courtesy of Martin Jechlinger

Fig. 5 3D Culture of MCF-10A mammary endothelial cells on 3D Culture MatrixTM RGF BME in Assay Medium with 2% BME and stained with Calcein-am

Cat. no

Description

Pack

3445-001-01

Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®

1 ml

3445-005-01

Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®

5 ml

3445-010-01

Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract, PathClear®

2 X 5 ml

Cultrex® 3D Culture Matrix Collagen I The 3D Culture Matrix™ rat collagen I may be used as a gel on which to grow cells or a medium additive, alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. Type I collagen is the major structural component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two α1(I) chains and one α2(I) chain that spontaneously forms a triple helix scaffold. This phenomenon can be exploited to promote cell attachment, growth, differentiation, migration, and tissue morphogenesis during development.

Fig.6 3D Culture of MCF-10A cells on 3D Culture Matrix Collagen I in Assay Media (DMEM, 2% Horse Serum, 20 mg/ml EGF, 500mg/ml Hydrocortisone, 100 mg/ml Cholera Toxin, 10mg/ml Insulin, and 1X Pen/Strep) with 2% BMe stained with SYBR® Green and Propidium Iodide.

Cat. no 3447-020-01

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Description Cultrex® 3D Culture Matrix Rat Collagen I

Pack 20 ml

Cultrex® 3D Culture Matrix Laminin I 3D Culture Matrix™ Mouse Laminin I may be used as a gel on which to grow cells, or as a media additive alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. To offer the most standardized Laminin I for use in 3D cultures, a special process is employed to provide material at a standard concentration of 6 mg/ml (by absorbance and extinction coefficient). Cat. no 3446-005-01

Description Cultrex® 3D Culture Matrix Laminin I

Pack 30 mg

Fig.7 Primary embryonic submandibular epithelium cultured in laminin-1 gel with FGF10 (200ng/ml). The Epithelia are cultured for either 24 or 48 hours in serum-free media and undergo branching morphogenesis in culture. Image courtesy of Matthew P. Hoffman. Patel, V.N., Knox, S.M., Likar, K.M., Lathrop, C.A., Hossain, R., Eftekhari, S., Elkins, M., Vlodasky, I., Whitelock, J.M. and Hoffman, M.P. Heparanase cleavage of heparin sulphate modulates FGF10 function during submandibular gland branching morphogenesis. Development, 2007, 134(23):4177-86

Cultrex® BME and Extracellular Matrix Proteins – Stem Cell Qualified Cultrex® Stem Cell Qualified Basement Membrane Extract (BME) is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumour. The extract gels at 37 °C to form a reconstituted basement membrane. It is mainly comprised of laminin, collagen IV, entactin, and heparin sulfate proteoglycan. BME typically ranges in protein concentrations from 14 to 16 mg/ml. Cultrex® Stem Cell Qualified BME has been shown to provide an effective feeder-free surface for the attachment of and maintenance of human embryonic stem cells in a pluripotent state, thereby enabling its use for growth promotion or for study of stem cell differentiation. Cat. no

Description

Pack

3434-005-02

Cultrex® Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract, PathClear®

5 ml

3420-001-03

Cultrex® Stem Cell Qualified Human Fibronectin, PathClear®

1 mg

3415-001-03

Cultrex® Stem Cell Qualified Human Basement Membrane Extract, PathClear®

1 ml

3421-001-03

Cultrex® Stem Cell Qualified Human Vitronectin, PathClear®

200 µg

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Individual Extracellular Matrix Proteins and Solutions • •

Variety of collagen sources Rat collagen supplied at high concentration

Cultrex® Mouse Laminin I, PathClear® Cat. no

Description

3400-010-02 Cultrex® Mouse Laminin I, PathClear®

Pack 1 mg

3446-005-01 Cultrex® 3D Culture Matrix 30 mg Laminin I

Fig.8 Laminin I promotes adhesion of MG63 cells. Laminin I was diluted to 5, 10 and 20 µg/ml and 100µl of each solution was added to six wells each of a 96 well plate and incubated overnight at 37◦ C. The coating solution was then aspirated , and wells were blocked for nonspecific binding with a 2$ BSA solution. MG63 cells were labelled with calcein am, harvested, and seeded at 15,000 cells/ well. The cells were allowed to attach for one hour and 15 minutes. Total fluorescence again. The percent adhesion was calculated from fluorescence of adhered cells divided by the fluorescence of total cells loaded for each well.

This highly purified preparation of mouse laminin I also increases cell adhesion, migration, proliferation, and differentiation. It is also composed of α1β1γ1 chains with a total Mr of 800,000 and is used for coating culture dishes.

Cultrex® Poly-L-Lysine This highly positively charged amino acid chain is commonly used as a coating agent to promote cell adhesion in culture. This solution is provided ready to use at 0.01% and contains polymers in the 70,000– 150,000 kDa range. Cat. no 3438-100-01

Description Cultrex® Poly-L-Lysine

Pack 100 ml

Cultrex® Poly-D-Lysine Poly-D-Lysine, a highly positively charged synthetic amino acid chain, is commonly used as a coating agent to promote cell adhesion in culture on TCT plastic or glass surfaces. Moreover, poly-D-Lysine is resistant to enzymatic degradation and promotes the proliferation and differentiation of a variety of neuronal cell lines. This solution is provided ready to use at 0.01% and contains polymers in the 70,000150,000 kDa range.

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Fig.9 Attachment and spreading of PC-12 cells rat adrenal gland pheochromocytoma grown on a plate coated with 0.01% Poly-L-Lysine.

Cultrex® Collagen I Type I collagen is the major structural component of extracellular matrices found in connective tissue and internal organs, but is most prevalent in the dermis, tendons, and bone. It is a 300 kDa molecule composed of two alpha1(I) chains and one alpha2(I) chain that spontaneously forms a triple helix scaffold at a neutral pH and 37°C. This phenomenon can be exploited to promote cell attachment, proliferation, differentiation, migration, and tissue morphogenesis during development. The 3D Culture Matrix™ Rat Collagen I may be used as a gel on which to grow cells or a medium additive, alone or in concert with other basement membrane components, to study cellular growth and differentiation in three dimensions in vitro. Cat. no

Description

Pack

3440-005-01

Cultrex® Rat Collagen I (5 mg/ml)

1 ml

3440-100-01

Cultrex® Rat Collagen I (5 mg/ml)

20 ml

3442-005-01

Cultrex® Collagen I (Bovine)

1 ml

3442-050-01

Cultrex® Collagen I (Bovine)

50 mg

3447-020-01

Cultrex® 3D Culture Matrix Rat Collagen I

20 ml

Fig. 10 MCF10A cells grown in collagen I gel and stained with Hoechst and anti-b-catenin antibody to visualize cell boundaries. Image courtesy of J. Pantanen & J. Klefstrom, Univeristy of Helsinki. Partanem, J.I., Makela, T.P. and Klefstrom, J.2007. Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization. PNAS, 104: 14694-14699

Cultrex® Mouse Collagen IV Collagen IV is the primary collagen found in the basement membranes, which underlie epithelial and endothelial cells and surround muscle, fat and nerve cells. Collagen IV can be used for coating of tissue culture surfaces to promote cell attachment and proliferation and to study its effects on cell behaviour. Cat. no 3410-010-01

Description Cultrex® Mouse Collagen IV

Pack 1 mg

Functional Attributes of ECM Component Proteins and Reagents Collagen IV

The wells within 20µg/ml Cultrex® collagen IV samples must exhibit cell attachment of equal to or greater than 25% whereas uncoated wells must have less than 10% attachment to pass quality control.

Rat Collagen I

Gelled rat collagen I should support a formation of network-like structures of SVEC4-10 cells compared to the cells adherent to the uncoated plastic surfaces only. The wells with the 20µg/ml Cultrex® Collagen I sample must exhibit cell attachment of equal to or greater than 40% whereas uncoated wells must have less than 10% attachment to pass quality control.

Bovine Collagen I

The wells with the 20µg/ml Cultrex® collagen I sample must exhibit cell attachment of equal to or greater than 40% whereas uncoated well must have less than 10% attachment to pass quality control.

Poly-L-Lysine

The wells with the 0.01% Cultrex® poly-L-Lysine sample must exhibit cell attachment of equal to or greater than 10% whereas uncoated wells must have less than 5% attachment to pass quality control.

Poly-D-Lysine

The wells with the 0.01% Cultrex® poly-D-Lysine sample must exhibit cell attachment of equal to or greater than 10% whereas uncoated wells must have less than 5% attachment to pass quality control.

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Cultrex® Human Fibronectin, PathClear® Fibronectin is an extracellular matrix protein that is found abundantly in blood and connective tissues. Its expression is associated with the epithelial to mesenchymal transition of metastatic cells, including tumour cells with stem cell-like properties. Fibronectin performs essential functions in collagen fibrillogenesis, as either a general cell adhesion molecule or a modulator in binding between cell surfaces and the extracellular matrix. Fibronectin matrix assembly is essential for normal vertebrate development and apparently contributes to the generation of tumour metastases by supporting the establishment and persistence of premetastatic niches. Fibronectin is secreted as a disulfide-linked dimer of 230-270 kDa, comprised of three types of repeating modules that mediate interactions with extracellular matrix components (including fibronectin itself), and cells via integrins and other fibronectin receptors. Thus, fibronectin can be used for coating tissue culture surfaces or as a medium additive to promote cell adhesion and proliferation; it is 0.2 μm sterile filtered. Cat. no 3420-001-01

Description Cultrex® Human Fibronectin, PathClear®

Pack 1 mg

Cultrex® Human Vitronectin, PathClear® Vitronectin is an extracellular, soluble, disulfide-linked dimer, composed of a 75 kDa and a 65 kDa peptide chain with a total molecular weight of 140 kDa. Vitronectin is a major plasma glycoprotein that promotes cellular adhesion and spreading. It also inhibits the membrane damaging effect of the terminal cytolytic complement pathway and binds to several serpin serine protease inhibitors. Vitronectin, along with collagen IV, fibronectin, and laminin can support robust, long term proliferation of undifferentiated human embryonic stem cells. Vitronectin can be used for coating tissue culture surfaces to promote cell adhesion, proliferation and differentiation, or as an additive for serum-free media. Vitronectin is purified from human plasma and is 0.2 μm sterile filtered. Cat. no

Description

Pack

3421-001-01

Cultrex® Human Vitronectin, PathClear®

3422-001-01

Cultrex® Human Vitronectin, Nucleic Acids Reduced, PathClear® 50 µg

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50 µg

Artificial Scaffolds & Matrices Naturally occurring hydrogels are an excellent tool to recapitulate the extracellular environment. However, they are verycomplex in composition and lack specificity to a particular tissue. As a result, several other solutions to the creation of physiologically relevant environment were developed. The one solution involves replacing EHS-based materials with alginate, a naturally occurring hydrogel which is inert. An entirely artificial solution is Alvetex®, a porous slab made of tissue culture plastic. This enables seamless transition from 2D to 3D environments with several advantages: very stable scaffold for cell to attach, grow and differentiate on; and ability to multiplex several cell types by combining slabs in various co-culture combinations. Alvetex® STRATA is a new generation of this technology extending it to tissue slices and embryonic bodies. MAPTrixTM bio-mimetics represent another approach to supplying extracellular matrix (and growth factor) signals. MAPTrixTM are recombinant mussel adhesion proteins containing one or two epitopes originating from micro-environmental proteins of interest. This represents a xeno-free approach to supplying these signals without batch to batch variation. There are also several pre-made MAPTrixTM arrays to screen for various extracellular protein requirements. There is great potential in combining this technology with lowattachment or Alvetex®-based solutions.

Alvetex® Scaffold Alvetex® is a highly porous polystyrene scaffold designed for 3D cell culture. Cells grown in Alvetex® form a tissue-like structure that enables them to function in a more physiologically relevant manner. Cells maintain their in vivo morphology, behaviour and responsiveness within an in vitro model system. Traditionally, cultured cells normally grow on treated-polystyrene 2D surfaces as in standard cell culture plastic-ware. Alvetex® presents cells with the equivalent growth substrate but in a 3D format. These materials are readily adaptable to different types of existing tissue culture plastic-ware (e.g. multi-well plates, well inserts). The culture device is pre-fabricated, sterile, is ready to use off-the-shelf and can be handled in a similar manner as standard 2D plastic-ware. There are distinct advantages in using Alvetex® over existing 3D culture products which are technically more difficult to use, have a finite shelf life and are expensive. Importantly, Alvetex® can be used for routine 3D cell culture, as an inert plastic. Alvetex® is a 3D culture product which can be treated in the same manner as traditional 2D cell culture plastic. The scaffold can be plasma treated without any detrimental effect to its structure. Alvetex® is sterilised using gamma radiation. In other words, its manufacture is compatible with standard culture-ware production. Furthermore, the use of polystyrene as a cell growth substrate is well accepted and recognised. The Alvetex®Scaffold 96-well plate is a high-throughput format suitable for drug discovery. Plates have black walls and a clear plastic base, with Alvetex®Scaffold at the bottom of each well, thereby allowing direct luminesce read-outs, such as cell proliferation and viability. MAIN BENEFITS Major cell types already used in Alvetex®Scaffold • • • • •

READ OUTS

Cancer: breast, colon, glioblastoma, lung and prostate Liver: including primary and liver carcinomas Skin: keratinocytes and full thickness models Human ES cells, mesenchymal stem cells, iPS and adipose tissue-derived stem cell (ASC) Other commonly used cells including 3T3, CHO, HUVECs and MDCK

• • • • •

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Alvetex® Scaffold allows many types of analysis once the 3D culture is complete: Imaging: histology, immunohistochemistry, confocal and electron microscopy Easy retrieval of DNA, RNA and protein for gene and protein expression analysis Simple biochemical analysis (viability /proliferation) Analysis of protein secretion media

SELECTED APPLICATIONS Cytotoxicity of MCF-7 cells to Tamoxifen in 2D compared to 3D culture on Alvetex ®

Cytotoxicity of HCT116 cells to 5-FU in 2D compared to 3D culture on Alvetex ®

Fig. 12 MCF-7 cancer cells were cultured on conventional 2D plastic (blue) or in 3D on Alvetex® (red) for 3 days prior to either (A) 24h or (B) 72h treatment with Tamoxifen. Percentage viability data from 3 sample replicates are shown (mean ± SD). (C) Brightfield micrographs showing morphology of MCF-7 cells grown for 3 days in 3D on Alvetex® prior to treatment for 24h with increasing concentrations of Tamoxifen.

Fig . 13 1,500 HCT116 cells – cultured 4 days – 5FU titrated in DMSO – added for 3 days

Function and Responsiveness of 3D Cultured Liver Cells in Alvetex®

Fig.14 Rat hepatocytes were cultured in 2D (grey bars) and 3D in Alvetex® Scaffold (black bars) for 48 or 72 hours after induction and assay of different CYP isoforms. Basal enzyme activities are also shown. Culture in Alvetex® Scaffold resulted in enhanced inducible levels of all three enzymes tested (CYP-1A2, -2B1, -3A2)

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Function and responsiveness of 3D cultured liver cells in Alvetex®

Fig. 15 Superior cell viability and albumin production of HepG2 cells grown in 3D Alvetex® Scaffold

Fig. 16 Co-cultures of glial cells (U118-MG) and stem cellderived neurons were set up to study cellular interactions and the effect of glial cells on neuritogenesis.

Fig. 17 Co-culture of Caco-2 cells and CCD-18 fibroblasts separated by a Collagen I layer in Alvetex®Scaffold. Low (A) and high (B) magnification brightfield micrographs of an even monolayer of Caco-2 cells form at the collagen-coated top surface of Alvetex®Scaffold, with CCD-18Co fibroblasts underneath the same collagen layer and distributed throughout the depth of Alvetex®Scaffold. CCD-18Co fibroblasts were grown on 22 mm diameter Alvetex®Scaffold discs presented in 6-well insert in 6-well plate format for 14 days prior to layering with Collagen I and seeding of Caco-2 cells. Co-cultures were grown for a further 5 days, after which they were fixed, embedded in paraffin wax, sectioned (10 μm) and counterstained with haematoxylin and eosin. Scale bars: 200 μm (A) and 50 μm (B).

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Co-culture and perfusion solutions using Alvetex® Modular & Flexible Alvetex® Co-culture Platform Technology Cell type A growing in 3D within Alvetex® Cell type B growing in 3D within Alvetex®

Different cell types cultured together within the same scaffold

Two independent 3D columns. Contact is via medium only and inter-culture communication via paracrine factors

MAIN BENEFITS • • • •

Straight-forward combination of co-culture in 3D Cells can be directly or in-directly co-cultured Flexibility in timeline of co-culture: cell types can be added and removed at any time Flow can be introduced into co-culture by using specially designed perfusion chambers

SELECTED APPLICATIONS Studying interactions between neurons and glia in co-culture models using Alvetex® Scaffold Fig.18 Studying interactions between neurons and glia in co-culture models using Alvetex® Scaffold. Co-cultures of glial cells (U118-MG) and stem cell-derived neurons were set up to study cellular interactions and the effect of glial cells on neuritogenesis. First, 1 million U118-MG glial cells were seeded onto 10µg/ml laminin and poly-D-lysine coated Alvetex® Scaffold and incubated for 15 min prior to the addition of neurospheres for a further 15 min. Co-cultures were incubated for 10 days and fixed in 4% PFA prior to imaging as before. Bright field micrographs showing low (A) and high (B) magnification images of neurosphere and glial co-cultures on Alvetex® Scaffold. Confocal imaging of neurospheres from above showed neurons producing neurites in the absence of U118MG cells (C, control) and suppression of neuritogenesis in the presence of the glial cells (D, plus U118-MG). Cells were stained with TuJ1 (green) and DAPI (blue) to show neurons and cell nuclei, respectively. Note the uniform distribution of glia cells (D, DAPI stained nuclei) and how neurites tend to wrap around the neurosphere avoiding contact with the U118-MG cells (D).

17 www.amsbio.com

Fig. 20 Setting up different culture systems within the wells. A) Well inserts of different types can be fitted into the plate, including Alvetex ® well inserts for 3D culture. Alvetex® well inserts have windows in their side wall to enable the flow of medium above and below the culture. B) 3D cultures can also be established in the base of the well and media slowly perfused above. This arrangement is compatible with both hydrogel and scaffold-based 3D technologies. C) Conventional 2D culture in the base of the well where a monolayer of cells is established and perfused with culture medium. Note: It is also possible to place tissue fragments in a well insert or base of the well and perfuse media over them for long-term maintenance in culture.

Fig. 19 Photograph showing Reinnervate Perfusion Plate set-up and connected to peristaltic pump. The plate has been raised by approximately 5 cm ( place an empty pipette tip box). Note the difference in the tube diameters: Media IN 1.6 mm and Media OUT 2.4 mm. The example shown includes four Alvetex® well inserts within the plate with incubation medium.

ORDERING INFORMATION Cat. no AMS. AVP010-2

Description

Pack

Alvetex® Scaffold 384-well plate

2x 384-well plates

AMS. AVP010-10 AMS. AVP011-2

10x 384-well plates Alvetex® Perfusion plates

2x Alvetex® perfusion plates and luer locks

AMS. AVP011-10

10x Alvetex® perfusion plates and luer locks

AMS. AVP-KIT-3

2x perfusion plates and luer locks 12x Alvetex® 6-well inserts

AMS. AVP-KIT-4

2x perfusion plates and luer locks 12x Alvetex® 12-well inserts

AMS. AVP-KIT-5

Perfusion plates with Alvetex® well inserts

AMS. AVP-KIT-6

5x perfusion plates and luer locks 48x Alvetex® 6-well inserts 5x perfusion plates and luer locks 48x Alvetex® 12-well inserts

18 www.amsbio.com

Alvetex® STRATA Alvetex® Strata, which is a second generation of porous material, primarily designed to support the growth of cells and intact tissues on the surface of the membrane. Alvetex® Strata is a highly porous membrane presented in a well insert format. This product has multiple applications, including the ability to stably support intact viable tissues during cell culture. subsequent analysis. At first glance, the structure of Alvetex® Strata appears similar to Alvetex® Scaffold: both products are made from the same crossedlinked polystyrene; both membranes are 200 microns thick; both are presented in our custom 6-well and 12-well inserts; and both come blister packed, sterile, ready for use. However, the difference between these two materials concerns their fine structure and architecture. Both materials are highly porous and comprised of voids and interconnecting pores: in Alvetex® Strata the voids and pores are significantly smaller (average 13 and 5 micron diameter, respectively)compared to those in Alvetex® Scaffold. READ OUTS

MAIN BENEFITS • • •

•

Enhanced porosity for improved nutritional support from the medium Modified surface topography to improved tissue attachment Versatility for co-culture and construction of advanced in vitro models

• •

SELECTED APPLICATIONS • •

Alvetex® Strata allows many types of analysis once the 3D culture is complete, including: Imaging : histology, immunohistochemistry, confocal and electron microscopy Easy retrieval of DNA, RNA and protein for gene and protein expression analysis Simple biochemical analysis (viability / proliferation) Analysis of protein secretion into culture media

Fig. 22 Immunocytochemical analysis of a human embryonic stem cell-derived embryoid body maintained for 7 days on an Alvetex Strata membrane presented in a 6-well insert. Staining for markers of the germ layer indicated the differentiation of ectodermal (B, TUJ1), tissues. DAPI staining showed the location of the cell nuclei. In some instances a few cells migrated into porous scaffold matrix that appeared to be mostly mesodermal in nature. Scale bars: 200 μm

ORDERING INFORMATION

Fig. 21 Time lapse imaging of spinal cord tissue slice and demonstration of tissue slippage. Images show the same field of view captured at time zero (A,C) and after 24 hours recording (B,D) for tissue slices maintained on either the Millicell (A,B) or Alvetex® Strata (C,D) porous membranes. Note how the cellular information captured in the field of view from the sample maintained on the Millicell membrane has all but slipped out of frame during the 24 hour period. In contrast, samples maintained on Alvetex® Strata remained almost completely static. The red arrows show the direction and extent of tissue slippage observed. Scale bar: 100 microns. (Images courtesy of Kieran McDermott, University of Cork.)

Cat. no AMS.STP004-12

Description

Pack

Alvetex® Strata 6 well inserts

12x strata 6-well inserts

AMS.STP004-48 AMS.STP005-12 AMS.STP005-48 19

www.amsbio.com

48x strata 6-well inserts Alvetex® Strata 12 well inserts

12x strata 12well inserts 48x strata 12well inserts

SELECTED APPLICATIONS

Alvetex®Strata enables the continual propagation of cells in 3D and maintenance of enhanced 3D structure and function.

Fig. 23 Passaging pluripotent stem cells in 3D results in their enhanced growth and differentiation when subsequently cultured as 3D suspended cell aggregates. TERA2.cl.SP12 cells were propagated for 4-1 0 passages in 2D or 3D culture and then cultured in suspension to form aggregates. Cells maintained in 3D for 6-10 passages formed significantly larger diameter aggregates compared to cells continually propagated in 2D culture (A). Data represent mean, ±SEM, n=10, **p=0.01, ***p=0.001. Aggregates were immuno-stained for the neural maker TUJ-1, and the epithelial marker cytokeratin-8 (B). Cells passaged in 3D resulted in aggregates with greater cellular heterogeneity including intense TUJ-1 staining and areas with lower cell density expressing cytokeratin-8. Cells propagated in 2D culture formed smaller aggregates that were primarily TUJ-1 positive. Scale bars: 100 μm

20 www.amsbio.com

ORDERING INFORMATION Cat. no AMS.AVP004-32

Description Alvetex® Scaffold 6-well inserts for 3D Culture

AMS.AVP004-48 AMS.AVP005 –34

6x 6-well insert 48x 12-well insert

Alvetex® Scaffold 12-well inserts for 3D culture

AMS.AVP005 –48 AMS.AVP002

Pack

12x 12 well insert 48x 12-well insert

Alvetex® Scaffold 12-well plate for 3D Culture

1x 12-well plate

AMS.AVP002-10

10x 12-well plate

AMS.AVP002-80

80x 12-well plate

AMS.AVP002-320

320x 12-well plate

AMS.AVP006

Alvetex® Scaffold 24-well plate for 3D Culture

1x 24-well plate

AMS.AVP006 –10

10x 24-well plate

AMS.AVP006 –80

80x 24-well plate

AMS.AVP006 –320

320x 24-well plate

AMS.AVP009

Alvetex® Scaffold 96-well plate for 3D culture

1x 96-well plate

AMS.AVP009 –10

10x9 6-well plate

AMS.AVP009 –80

80x 96-well plate

AMS.AVP009 –320

320x 96-well plate

Cat. no

Description

Pack

AMS.AVP-KIT-2

Alvetex® Well Insert Starter Kit

Contains 6x6-well inserts, 6x12well inserts and 1 deep petri dish

AMS.AVP-KIT-1

Alvetex® Plate Starter Kit

Contains: 1x12-well 1x24-well 1x96-well plates

AMS.AVP015-2

Alvetex® Well insert holders and petri dishes for 3D culture

2x insert holders 2x petri dishes

21 www.amsbio.com

Alginate Alginate, an anionic polysaccharide derived from cell walls of brown algae, forms a gel in the presence of calcium and liquefies to a solution upon addition of a calcium chelating agent. Alginate hydrogel has been a choice for 3D cell culture because cultured cells can be easily harvested MAIN BENEFITS

READ OUTS

The Alginate 3D Cell Culture Kit is a convenient, easy-tomanufacture kit optimized to produce alginate gel beads. This product has been used to develop successful 3D cell culture systems for a range of different cells types including tumour cells and chondrocytes.

• • • • •

Viability/cytotoxicity Cell signalling and communication Tissue regeneration Imaging: immunofluorescence, confocal microscopy and immunohistochemistry Retrieval of DNA, and RNA for gene analysis

SELECTED APPLICATIONS

Fig.24 HepG2 cells, a human liver cell line, cultured in alginate beads fro 9 days (left: low magnification, right: high magnification)

ORDERING INFORMATION Cat. no AMS.CSR-ABC-KIT

Description Alginate 3D Cell Culture Kit

Fig. 25 Growth curve of HepG2 cellscultured in alginate beads (Mean±SD, n+3). HepG2 cells were cultured in alginate beads (5x 105 cells/mL, 10 beads/ well, DMEM containing 10% FBS) . At end of the culture, the alginate beads were dissolved with sodium citrate solution and the cell pallet was digested with Pronase solution (1mg/mL). The DNA content of the digested sample was determined using the Hoechst 33258 fluorescent dye method.

Pack 1 x Alginate 3D Cell Culture Kit

22 www.amsbio.com

MAPTrix™ - Recombinant Animal Free Extracellular Matrix Mussel Adhesive Protein based matrix (MAPTrixTM) recombinant extracellular matrix (ECM) acts as bio-mimetic for traditional basement membrane extracts. MAPTrixTM replaces traditional ECM with genetically incorporated bioactive peptides (recognition peptides) that provide an environment for the maintenance of cells under serum and feeder-free conditions.

P R

S D G R G

I S L Q V Q L

MAPtrix TM ECM coated Surface

MAPTrix™ ECM offers flexible prefabricated building blocks as a tool to engineer extracellular microenvironment MAPTrix™ technology for extracellular matrix (ECM) based coatings or surface modification is simple, convenient and highly reproducible. You can readily engineer a synthetic ECM surface that binds to adhesion receptors such as integrins and promote cell adhesion and spreading. MAPTrixTM utilises mussel adhesive protein to create the first combinatorial synthetic ECM library for engineering integrin specific surfaces. These surfaces mimic the native extracellular environment. Mussel adhesive protein is highly desirable for use in a variety of biological and medical applications due to its strong wet adhesive, non toxic, biodegradable and low immunogenicity properties.

Bioactive peptide genetically fused to MAP

Mussel Mussel Adhesive adhesive Protein protein

E.coli

Bioactive Peptide

Adhesive Domain

MAPTrix™ biomimetics are already used in: • Stem cell technology • Tissue engineering scaffolds • Drug delivery • Cell surface modification • Coating of medical devices

Bioactive Domain

READ OUTS • • • • •

23 www.amsbio.com

Defined media conditions Defined adhesion conditions Adhesion assays Proliferation assays Other ECM-dependent functional assays

MAIN BENEFITS • • • • • • • •

Biochemically-defined & animal-free Reproducible & reliable protein coating Low cost Ready to use Improved cell morphology and cell proliferation Adhere to USP guidelines FDA recommendations compliant Eliminates risk of animal or viral infectious agents in cell cultures

SELECTED APPLICATIONS

Fig. 26 Mesenchymal stem cells from male mouse adipose were encapsulated and cultured in hydrogel containing MAPTrix™ Collagen type I mimetic for 7 days. The encapsulated MSCs were then transplanted to the abdominal cavity of a female mouse for one month before analysis (If Y chromosome is detected in the abdominal cavity tissue, it indicates that the tissue came from the encapsulated stem cells).

MAPTrixTM Mediated Cell Adhesion and Growth MAPTrixTM Laminin

GibcoTM Laminin

Sigma Poly-L-lysine

Focal Adhesion

Actin Formation

Axons and dendritic processes growth

Fig.27 MAPTrixTM laminin bio-mimetic is comparable to native laminin and poly-L-lysine in supporting neuronal adhesion and outgrowth. (A) Primary nerve cells form similar focal adhesions on both MAPTrixTM bio-mimetic (left panels) and native laminin (middle panels). (B) Hippocampal neuronal cells outgrowth is also equally supported by MAPTrixTM and native laminin. Both laminins were superior to ploy-L-lysine (right panels) in supporting adhesion and axon dendritic growth.

24 www.amsbio.com

Fibronectin Derived Peptides

Fibronectin (FN) is a high molecular weight glycoprotein that consists of three types of repeating amino acid units: type I, type II, and type III repeats. The structure of fibronectin depends on whether it is secreted in plasma or synthesized by resident cells. Cellular fibronectin contains the alternatively spliced extra domain A and/or extra domain B. In addition, a third alternatively spliced domain, the IIICS domain (for rodents: the V-region), can be included, but regulations for its inclusion have not been fully discovered yet. Fibronectin naturally exists as a dimer, consisting of two nearly identical monomers. Two regions in each fibronectin subunit possess cell binding activity: III9-10 and III14-V. The primary receptor for adhesion to fibronectin commonly involves the RGD motif of repeat III10 through integrins such as α5β1. However, this integrin-ligand interaction is only sufficient for cell attachment and spreading. Additional signalling through the cell surface proteoglycan such as syndecan-4 is required for focal adhesion formation and rearrangement of the actin cytoskeleton into bundled stress fibres. This binding occurs primarily via the Hep II domain (containing the FN type III repeats 12-14) in the C-terminal region of fibronectin.

Domain

Peptide Motif

Cat. No *

Domain

Peptide Motif

Cat. No *

Type III-5

KLDAPT

16103X

Type III-13

ATETTITIS

16111X

Type III CS-1

PHSRN

16104X

FN-C/H-V

WQPPRARI

16116X

Type III-10

RGD

16105X

FN-C/H-II

KNNQKSEPLIGRKKT

16119X

Type III-10

GRGDSP

16107X

Type III CS-1

EILDVPST

16120X

FN-C/H-III

YRVRVTPKEKTGPMKE

16109X

Type III CS-5

REDV

16124X

FN-C/H-1V

SPPRRARVT

16110X

Type III

PHSRN-RGDSP

16125X

KEY TO CATALOG NUMBERING Please see next page Collagen Derived Peptides

Collagens serve as scaffolds for the attachment of cells and matrix proteins. Collagen is the major insoluble fibrous protein in the extracellular matrix and in connective tissue. In fact, it is the single most abundant protein in the animal kingdom. There are at least 16 types of collagen, but 80 – 90 percent of the collagen in the body consists of types I, II, and III. These collagen molecules form long thin fibrils. Type IV, in contrast, forms a two-dimensional reticulum; several other types associate with fibril-type collagens, linking them to each other or to other matrix components. The various collagens and the structures they form help tissues withstand stretching. Collagens are also highly biologically active, with many other ligands. For example, collagens provide integrin- and heparin-binding motifs. α2β1 integrin recognizes GXO/SGER such as GFPGER or GFOGER for endothelial cell binding / activation and angiogenesis. Integrin binding sites for αvβ3 have antitumor activity, and may inhibit the activation of human neutrophil or the proliferation of capillary endothelial cells. Integrin binding sites in the NC1 domains have anti-angiogeneic properties mediated by the α1β1 or αvβ3 integrin binding. 25 www.amsbio.com

Collagen Derived Peptides (Continued) Domain

Peptide Motif

Cat. No *

Domain

Peptide Motif

Cat. No *

Type I α1

GLPGER

16501X

Type IV α1

TAGSCLRKFSTM

16621X

Type I α1

KGHRGF

16502X

Type IV α1

GEFYFDLRLKGDK

16623X

Type I α1

GFPGER

16104X

Type IV α3

TAIPSCPEGTVPLYS

16631X

Type I α1

DGEA

16506X

Type IV α3

TDIPPCPHGWISLWK

16632X

Type I α1

GPAGKDGEAGAQG

16107X

Type IV α3

ISRCQVCMKKRH

16635X

Type I α1

GTPGPQGIAGQRDVV

16512X

Laminin Derived Peptides

Laminins (heterotrimers composed of α, β, and γ chains), are multifunctional glycoproteins present in basement membranes. Integrins, dystroglycan, syndecans, and several other cell surface molecules are cellular receptors for laminins. The globular domains located in the N- and C-terminus of the laminin α chains are critical for interactions with the cellular receptors. Integrin α6β1 binds to most of the laminin isoforms. Integrin α3β1 interacts with laminin-5 and -10/11 more specifically than the other isoforms. Integrins α1β1, α2β1, and α7β1 show binding activity to laminin-1 and -2. Interaction of integrin α6β4 with laminin-5 forms hemidesmosomes in the skin. α-dystroglycan strongly binds to the laminin α1 and α2 chains and moderately interacts with the α5 chain. Domain

Peptide Motif

Cat. No *

Domain

Peptide Motif

Cat. No *

α1 chain

RQVFQVAYIIIKA

16204X

α5 chain

GIIFFL

16369X

α1 chain

IKVAV

16224X

β1 chain

RYVVLPR

16411X

α1 chain

AASIKVAVSADR

16225X

β1 chain

YIGSR

16414X

α1 chain

NRWHSIYITRFG

16226X

β1 chain

LGTIPG

16421X

α1 chain

TWYKIAFQRNRK

16229X

γ1 chain

KAFDITYVRLKF

16442X

α1 chain

RKRLQVQLSIRT

16232X

SETTVKYIFRLHE

16452X

α3 chain

PPFLMLLKGSTR

16288X

RNIAEIIKDI

16460X

α3 chain

KNSFMALYLSKGRLVFALG

16293X

γ1 chain

*KEY TO CATALOG NUMBERING Cat. No. ending with X =

Pack size+

1 mg protein, aqueous solution at 0.2mg/mL

2.5 mg protein, aqueous solution at 0.5mg/mL

5 mg protein, aqueous solution at 0.5mg/mL

10 mg protein, aqueous solution at 1mg/mL

26 www.amsbio.com

Additional Adhesion Peptides Some extracellular matrix components are adhesion-modulatory extracellular matrix proteins which interact with the main ECM components or integrins. These proteins include vitronectin, nidogen, Tenascin, and SIBLINGs (small integrin-binding ligand, N-linked glycoprotein) such as bone sialoprotein (BSP) and can also influence the cellular behaviour by regulating cell signalling (directly or indirectly). Cadherins are calcium-dependent cell adhesion proteins which are involved in many morphoregulatory processes including the establishment of tissue boundaries, tissue rearrangement, cell differentiation, and metastasis. The extracellular domain of E-cadherin tends to bind in a homophilic manner; although heterophilic binding does occur under certain conditions.The binding of extracellular cadherin is the basis for cell-cell adhesion, tends to be prevalent at adherin junctions and is structurally associated with actin bundles. Domain

Peptide Motif

Cat. No*

Domain

Peptide Motif

Cat. No*

Cadherin E-cadherin ECD1

SHAVSS

16701X

Nidogen G2

LNRQELFPFG

16811X

E-cadherin ECD1

LFSHAVSSNG

16702X

Nidogen G2

SIGFRGDGQTC

16812X

E-cadherin ECD1

ADTPPV

16703X

Tenascin-C

VAEIDGIEL

16831X

E-cadherin, Ca2+ binding

DQNDN

16706X

Tenascin-C

VFDNFVLK

16832X

N-cadherin, ECD1

HAVDI

16707X

Elastin

VGVAPG

16851X

N-cadherin ECD1

LRAHAVDING

16708X

Bone Sialoprotein (BSP)

KRSR

16901X

N-cadherin ECD1

LRAHAVDVNG

16709X

Bone Sialoprotein (BSP)

FHRRIKA

16902X

HVP

FRHRNRKGY

16801X

TTSWSQCSKS

16931X

HVP

KKQRFRHRNRKGYRSQ

16802X

CCN (connective growth factor) Fibrinogen

HHLGGAKQAGDV

16953X

Somatomedin B

RGDV

16803X

Vitronectin

*KEY TO CATALOG NUMBERING Cat. No. ending with X =

Pack size+

1 mg protein, aqueous solution at 0.2mg/mL

2.5 mg protein, aqueous solution at 0.5mg/mL

5 mg protein, aqueous solution at 0.5mg/mL

10 mg protein, aqueous solution at 1mg/mL

27 www.amsbio.com

Scaffold Free Solutions One of the most successful and popular approaches to creating physiologically relevant cell cultures has been scaffold-free solutions. At their simplest format, a small number of cells can be seeded on a low-attachment surface to induce the clustering of cells into spheroids. In most cases, several other signals will be required to generate the correct micro-environment: these can come from other cell types in the culture system, addition of extracellular matrices or any other reagents added. Lipidure-COAT plasticware is an excellent straight-forward low-attachment solution, such as for the formation of embryonic bodies. Other spheroid-based approaches are shown to quantify the proliferation and invasion of cancer cells. The formation of those spheroids can be assisted by specialised basement membrane extract to enhance the adhesion of the cells to each other. This ensures the ability to quantify the size of each spheroid in a 96-well plate and enhance the throughput of these assays. Addition of another specialised basement membrane extract enables invasion from the central spheroid, again enabling quantitative approach to this complex and important process in cancer biology.

Lipidure®- COAT Low-Attachment Solutions Spheroid cell culture is typically based on the spontaneous formation of an aggregate of cells in an environment where cell-cell interactions dominate over cell-substrate interaction. This can be achieved by using low-attachment cell culture conditions. Lipidure®-COAT plates and dishes are a top of the range solution for spheroid formation, with the Lipidure coating providing a superior low-attachment solution for the formation of single spheroids in each well of multi-well plates. Using Lipidure®-COAT dishes, it is possible to undertake large scale assays to provide sufficient materials for applications such as Western blotting or gene expression microarrays. Lipidure®-COAT solutions have several advantages over other technologies: 1. Low-adhesion surface promotes cell aggregation & spheroid formation. 2. Uses MPC Polymer: a biocompatible polymer containing Phosphoryl Choline (which is found in cell membranes) 3. Completely synthetic, containing no substances of biological origin. Cell

Human Cell Surface

CH3

C=O

Suppression of Cell adhesion

CH2—C

CH2— C O-

CH3

C=O

OCH2 CH2 OPOCH2 CH2 N—CH3 O

CH3

Substrate

R n

Phosphorylcholine

MAIN BENEFITS • Easy to handle • Excellent reproducible results • Providing a superior low-attachment solution • Compatible with a variety of cell-based assays • Multiple formats for simple assay up-scaling

Artificial Cell Surface

READ OUTS • • •

28 www.amsbio.com

Bright field imaging (clear plates) Fluorescence (black plates) Luminescence (white plates)

SELECTED APPLICATIONS Low Cell Binding Property of Lipidure®- COAT

Fig. 28 NIH 3T3 (Fibroblast, mouse) spheroids grown for 4 days on Lipidure®-COAT Multi-Dish A-6MD and uncoated plat; then washed with PBS and low cell binding properties estimated

Superior Formation of Single Spheroid on U-Bottom Plate

Fig. 29 A single spheroid is generated in the Lipidure-COAT well, while numerous satellite spheroids are found in the competitor’s plates. This phenomenon shows that a lot of cells adhered to the competitor’s “low-adhesion” surface.

Seeding Number ( cells/well)

Diameter of Spheroid (µm) Lipidure-COAT

Competitor

1,000

185±12

161±9

10,000

423±7

323±46

Generation of embryonic bodies in low-attachment environment Single Spheroid

STEP 1 Monolayer culture

STEP 2 Plate single cells

STEP 3 Cell aggregation

Fig. 30 A single embryonic body spheroid is formed in each U-bottom well within 5 days

29 www.amsbio.com

STEP 4 Formation of Spheroid

Embryonic Body Formation

Diameter of EES 袖m

1000 800 600 400 200 0

1000

200

4000

Seeded cell density of mES cells (cells/well)

Fig. 31 Example of embryonic bodies formed using variable starting densities of the murine ES cell line 129SV on Lipidure速-COAT Plate A-U96

Fig. 32 Neurospheres formed by Rat Hippocampus Neuronal Cells by day 5 on Lipidure速- COAT Plate A-U96. Photo supplied by Dr Ljima, Kyusyu University

Anti-Cancer Drug Tests

Fig. 33 Spheroid formation and application in Anti-Cancer Drug tests Using Lipidure速-Coat. Hep G2 spheroids were formed for 5 days. Cell viability was determined by WST assay after further 2 days incubation with or without Mitomycin-C

30 www.amsbio.com

WELL SHAPES for 96-well plates Bottom shape can control size, position and density of spheroids

Flat Bottom

Cat. No

U-Bottom

V-Bottom

Product Name

Description

Pack

AMS.51011610

Lipidure-COAT Plate A-U96

96 wells (U-Bottom)

7 pieces

AMS.51011611

Lipidure-COAT Plate A-F96

96 wells (Flat Bottom)

7 pieces

AMS.51011612

Lipidure-COAT Plate A-V96

96 wells (V- Bottom)

7 pieces

AMS.51011617

Lipidure-COAT Multi-Dish A-6MD

6 well plate

7 pieces

AMS.51011618

Lipidure-COAT Multi-Dish A-12MD

12 well plate

7 pieces

AMS.51011619

Lipidure-COAT Multi-Dish A-24MD

24 well plate

7 pieces

AMS.51011614

Lipidure-COAT Dish A-60D

60mm Dish

20 pieces

AMS.51011615

Lipidure-COAT Dish A-90D

90mm Dish

20 pieces

Coating Polymer also available AMS.52000011GB1G

Lipidure-CM5206

White powder

AMS.52000012GB10G

Lipidure-CM5206

White powder

10 g

AMS.52000012GB100G Lipidure-CM5206

White powder

100 g

AMS.51011801

Lipidure-COAT

384 wells White

1 plate

AMS.51011802

Lipidure-COAT

384 wells Clear

1 plate

31 www.amsbio.com

Spheroid-based Assays Spheroid-based kits are the next step in the evolution of 3D culture. These 96-well kits utilise specially formulated extracellular matrix to enhance the generation of multicellular spheroid models for quantification of proliferation and invasion. While these assays were developed primarily for cancer cells, they could be used with other cell types with invasive properties such as endothelial cells. The 96-well spheroid formation plate are a flexible, standardized, high-throughput format to study cell proliferation and invasion. Cells are added to 3D Culture qualified specialized matrices for spheroid formation and invasion with no transfer steps required. This allows for simple and easy standardisation.

MAIN BENEFITS • • • • • •

READ OUTS

Similar morphology Formation of cell-cell bonds Decreased proliferation rates Increased cell survival Tumor dormancy Hypoxic core

• • • •

32 www.amsbio.com

Proliferation Viability Invasion Immunofluorescence

Proliferation / Viability AssaysQuantitate with Resazurin (Fluorescent)/ MTT (Colorimetric)

RFU

15,000 10,000 5,000 94 750 188 375 1,500

3,000

Spheroid Seeding Density (Cells/Well) Fig. 34 MDA-MB231 Spheroids after 7 days in culture

SELECTED APPLICATIONS

Fig. 35 Spheroid growth of MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37◦ C, 5% CO2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37◦ C, 5% CO2 . Spheroids were photographed every 24 hours, and images were analyzed using ImageJ software.

Fig. 36 Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cell/well in the presence of spheroid formation ECM and incubated for 72 hours at 37◦ C, 5% CO2 to induce spheorid formation. Spheroids were then treated with the coreesponding doses of Bleomycin and incubated 37◦ C, 5% CO2 for 96 hours. Spheroids were photographed after 96 hours, and images were analyzed using ImageJ software.

33 www.amsbio.com

SELECTED APPLICATIONS

CELL LIN E

Days Post Embedding 2

Fig. 37 Invasion of cancer cells out of MCTS and into the surrounding invasion matrix in a time-dependant manner. Cancer cells invade into invasion matrix composed of 10 mg/ml BME and 1gm/ml collagen I in response to cell culture medium containing 10% FBS; wells were photographed every 24 hours over a 96 hour period

Cat. No

Description

Pack

3500-096-K

Cultrex速 3D Spheroid Cell Invasion Assay

96 samples

3510-096-K

Cultrex速 3D Spheroid Fluorometric Proliferation/Viability Assay

96 samples

3511-096-K

Cultrex速 3D Spheroid Colorimetric Proliferation/Viability Assay

96 samples

34 www.amsbio.com

Cell Behaviour Assays

READ OUTS Cell behaviour assays cover a broad range of physiological functions: Cell adhesion

Cell adhesion is the binding of a cell to a surface, such as an extracellular matrix or another cell, using cell adhesion molecules. Cell adhesive interactions play important roles during many normal physiological processes such as embryonic development and wound repair, and also during the progression of diseases such as cancer. Cell adhesion is mediated by the specific interactions of cell surface receptors with extracellular glycoproteins. The bestcharacterized cell adhesion receptors are the integrins. Cellular adhesion through integrins links the cytoplasm of cells and is involved in signal transduction.

Cell migration

Cell migration is the movement of cells in response to a chemical stimulus; this is also known as chemotaxis. Traditionally, cell migration assays evaluate cell migration based on the cells’ ability to traverse an uncoated membrane with pores, in response to a chemotactic gradient. The cells must undergo cytoskeletal remodelling to fit into the pores and pull them through to the underside of the membrane. Alternatively, Oris assays quantify migration or invasion through extracellular proteins into the empty centre of each micro-plate well.

Cell invasion

Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Here, the membranes are coated with a layer of extracellular matrix proteins, and the cells must traverse this barrier through a combination of protein degradation and cellular locomotion.

Other assays in this section

The major variables associated with 3D culture are cell type, cell growth, composition of the physiological barrier, thickness of the physiological barrier, chemo-attractant that is used, and time of culture.

The quantification of cellular responses is the bedrock of cell biology. Assays here enable precise evaluation in physiological context of variety of cell types: • Adhesion, migration and invasion • Proliferation • Angiogenesis and vascular permeability

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MAPTrixTM Adhesion Arrays For many cell types, the exact requirements to generate a physiologically-relevant micro-environment are unknown. MAPTrixTM biomimetics are xeno-free solution based on mussel adhesion protein technology to provide micro-environmental signals (see also pp. 22-26). Using an array of MAPTrixTM arrays, cellular requirements can be screened. Below are standard arrays containing MAPTrixTM bio-mimetics for the most frequently used extracellular matrix proteins. Custom arrays for specific requirements – containing MAPTrixTM with biomimetics for extracellular proteins and/or growth factors are also available. MAPTrixTM arrays, provides a high-throughput platform to screen for the behaviour of cells under a variety of micro-environmental signals. This can be used to find which signals are required or study the different behaviour of cells in response to them. MAPTrixTM arrays can be used to select specific biomimetic to be incorporated into other environments such as cell culture media or as essential signal provided in addition to existing artificial or natural scaffolds. MAIN BENEFITS

READ OUTS

• •

• • •

• •

Easy read-out using any plate reader or imaging technology Flexible platform to study adhesion, proliferation or other cellular process depending on the micro-environment Easy comparison between micro-environmental signals – the recombinant MAPTrixTM backbone is identical between all wells MAPTrixTM found to be ideal on arrays can be bought for future use as standalone products for any assay or as part of defined media

Adhesion assays Proliferation assays Other ECM-dependent functional assays

Basic ECM mimetic screen ANN 901

Cat. No ANN901 ANN902 ANN903

Description

Pack

MAPTrix Screen for Cell Adhesion Assay, 24 different ECM mimetics coated 96 well plate (4 duplicates) TM

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2 plates per case 5 plates per case 10 plates per case

Fig. 38 Primary HUVEC cells were incubated on MAPTrix™ ECM arrays for 48 hours in serum free conditions. Cell counts were normalized against average cell counts on Non-coating (BD Falcon). Each bar represents the mean value of three wells

CultreCoat® Cell Adhesion Assays CultreCoat® Cell Adhesion Assays provide a simple, standardized, 96 well high throughput format for assessing factors that influence cell-matrix interactions. The black stripwell plate minimizes background, providing greater sensitivity and flexibility for the number of samples assessed. Calcein - AM labeling allows direct comparisons between the number of cells that are loaded and the number that adhere, providing a loading control and the percent cell adhesion based on the final fluorescence assessed for each well. Controls are provided for determining background and non-specific binding. The CultreCoat® Cell Adhesion Protein Array in one 96-well plate provides a format to assess factors that influence cell adhesion on BME, laminin, collagen I, collagen IV, fibronectin, and vitronectin. The black stripwell plate minimizes background, providing greater sensitivity, and affords flexibility for the number of samples assessed. The stripwell feature also allows multiple experiments to be conducted simultaneously using the same kit. Choice of matrix should correspond to the physiological micro-environment to be recapitulated. Basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils. Cat.No

Description

Pack

3490-096-K

CultreCoat® BME 96 Well Cell Adhesion Assay

96 tests

3491-096-K

CultreCoat® Laminin I 96 Well Cell Adhesion Assay

96 tests

3492-096-K

CultreCoat® Collagen I 96 Well Cell Adhesion Assay

96 tests

3493-096-K

CultreCoat® Collagen IV 96 Well Cell Adhesion Assay

96 tests

3494-096-K

CultreCoat® Fibronectin 96 Well Cell Adhesion Assay

96 tests

3495-096-K

CultreCoat® Vitronectin 96 Well Cell Adhesion Assay

96 tests

3496-096-K

CultreCoat® Adhesion Protein Array Kit

96 samples

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Cultrex® Cell Invasion Assays Cell Invasion Assays were created in an effort to accelerate the screening process for compounds that influence cellular degradation of and migration across extracellular matrices, which is a fundamental component of cellular processes such as angiogenesis, embryonic development, immune responses, and tumour cell metastasis. These assays offer a flexible, standardized, high-throughput format for quantitating the degree to which invasive cells penetrate a barrier consisting of basement membrane components in vitro in response to chemo-attractants and/or inhibiting compounds These assays employ a simplified Boyden chamber design with a polyethylene terephthalate (PET) membrane containing 8µ pores, which allow access from the input chamber (top) to the assay chamber (bottom) without dismantling the device. The assay chamber may be directly analysed in a 96-well plate reader, eliminating transfer steps that introduce additional variability to the assay. Cell invasion is quantified using calcein-acetoxymethyl ester (Calcein-AM), which is internalized by the cells, and cleaved by intracellular esterases to generate fluorescence signals. In conjunction with a standard curve, these signals may be used to quantitate the number of cells that have migrated or invaded – thereby eliminating the need for direct cell counting. Since different cell lines and different treatments can result in a wide range of invasive potentials, the permissiveness of each matrix may also be optimized to fit each experiment by adjusting the coating concentration. Cultrex® Cell Invasion Assays provide multiple formats for evaluation against different extracellular matrices and matrix components: laminin I, collagen I, collagen IV, and basement membrane extract (BME).

Cellsinvading invadingbarrier barrier Cells

Cells dissociated & fluorescence labelled

Fluorescence read directly from plates

Fig. 39 Illustration of the cell invasion protocol

Fig. 40 Different coating densities for the 96 Well BME Cell Invasion Chamber results in different invasion rates for invasive cell lines. HT-1080, human fibrosarcoma, and MDAMB-231, breast cancer, cell lines were serum starved for 16 hours and seeded at 25,000 cells per well in pre coated 8 µm chambers. Cells invaded in response to 10% FBS over a 24 hour period and were quantitated using Calcein-AM. Samples were in quadruplicate.

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CultreCoat® Cell Invasion Optimization System The CultreCoat® Optimization System is a flexible approach that allows you to choose the desired degree of cell invasion necessary for a specific project. Included in the system are a series of Boyden chambers coated at three distinct concentrations of BME and uncoated wells to compare invasion to basal migration. Relative to uncoated wells, cell invasion decreases as the concentration of the BME coating increases. This system provides an easy approach to determine the ideal coating density that provides the maximum signal-to-noise ratio and dynamic required for your study. Once the desired concentration is determined, BME coated plates can be ordered. The system is available in both 96-well and 24-well formats. Cat.No

Description

Pack

3455-024-K

Cultrex® 24-Well BME Cell Invasion Assay

24 tests

3455-096-K

Cultrex® 96-Well BME Cell Invasion Assay

96 tests

3456-024-K

Cultrex® 24-Well Laminin I Cell Invasion Assay

24 tests

3456-096-K

Cultrex® 96-Well Laminin I Cell Invasion Assay

96 tests

3457-024-K

Cultrex® 24-Well Collagen I Cell Invasion Assay

24 tests

3457-096-K

Cultrex® 96-Well Collagen I Cell Invasion Assay

96 tests

3458-024-K

Cultrex® 24-Well Collagen IV Cell Invasion Assay

24 tests

3458-096-K

Cultrex® 96-Well Collagen IV Cell Invasion Assay

96 tests

3480-024-K

CultreCoat® Original 24 BME Well Cell Invasion Assay

24 tests

3481-024-K

CultreCoat® 24-Well Low BME Cell Invasion Optimization Assay

24 tests

3481-096-K

CultreCoat® 96-Well Low BME Cell Invasion Assay

96 tests

3482-024-K

CultreCoat® 24-Well Medium BME Cell Invasion Optimization Assay

24 tests

3482-096-K

CultreCoat® 96-Well Medium BME Cell Invasion Optimization Assay

96 tests

3483-024-K

CultreCoat® 24-Well High BME Cell Invasion Optimization Assay

24 tests

3483-096-K

CultreCoat® 96-Well High BME Cell Invasion Optimization Assay

96 tests

3481-024-01

CultreCoat® 24-Well Low BME Cell Invasion Inserts with Plates

24 inserts

3484-024-01

CultreCoat® 24-Well BME Cell Invasion Optimization Inserts with Plates

24 inserts

Oris™ Cell Migration and Invasion Assays The migration of cells is known to occur during a vast array of both normal biological processes such as the development of an organism, immune response and wound healing or marking pathological events such as cancer metastasis, cardiovascular disease and arthritis. The need to better understand the process of cell migration has led to the development of improved methods of investigation including culturing methods, live cell trackers and detection methods. Improvements in both the investigative methods and throughput technologies will allow for rapid expansion of investigation in the area of cell migration research. OrisTM Pro Migration and Invasion Cell Assays are a reproducible, sensitive, and flexible assay that can be used to monitor cell migration (see figure overleaf). Formatted in a well plate, the assay uses non-toxic biocompatible gel (BGC) to form a cell-free zone. After seeding cells into the well plate, the BCG self-dissolves permitting cells to migrate or invade through collagen into the well centres. The OrisTM Pro Assays enables the use of automated liquid handling equipment for ell seeding and allows for unlimited access to wells from cell seeding through data readout. The OrisTM Pro Cell Migration Assay is designed to be used with any commercially available stain or labelling technique. Researchers can capture and quantify real-time and endpoint cell migration data using inverted microscopes. High content screening and high content imaging instruments.

39 www.amsbio.com

Cells seeds & adhere

Detection zone is created

Cells migrate or invade into detection zone

Analysis of cells in detection zone

MAIN BENEFITS • Enhanced Efficiency– Screen more compounds in a fully automatable 384-well format • Increased Reliability– Obtain robust and reproducible data from high content imaging/ high content screening (HCI/HCS) instrumentation • Generate More Useful Data– Use multiplexed staining to simultaneously measure cell movement, morphology and phenotypic changes SELECTED APPLICATIONS

Fig. 41 Dose response curves. MDA-MB-231/GFP and HT-1080 CT cell were imaged in the 96-well microplate format after treatment with CD in dose response format A) Image J analysis plots percent closure vs. inhibitor concentration while B) Gen5 analysis plots standard deviation vs. inhibitor concentration.

Fig. 42 Seeding density and incubation time optimization. Representative images from a well containing 50K HT-1080 cells per well in a 96-well plate format imaged kinetically every two hour for 12 hours.

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SELECTED APPLICATIONS

Fig. 43 Image analysing using imageJ to generate orthogonal view image. Z-stack images of HT-1080 cells was analysed using Image (NIH) software. Representative image shown above was taken at 2.5x magnification. Stacks of the image were taken at optimal Nyquist rate 21.39 µm. Image was processed to obtain orthogonal views of the XZ plane (bottom panel) an YZ plane (right panel) . The Z-lines were set at the zero plane for each well. Total height of Z-stacks is 1519 µm. An outline of the 2 mm diameter detection zone ( red dashed lines) serves as a reference point for cell invasion which has been superimposed onto all images accordingly.

Fig. 44 Effect of collagen overlay density as a function of time in cell invasion assay: Increasing the concentration of the collagen overlay restricts the invasion of HT-1080 cells. An OrisTM Invasion formed with HT-1080 cells overlaid with 1,2 or 3 mg/mL collagen and assayed at 0,24,48 and 72h. The results demonstrate an increase in cell invasion as a function of time. Lower levels of invasion were seen in the higher concentration collagen matrices. At 24h, cell movement into detection zone ( dashed vertical lines) was observed. At 48h and 72h, significant invasion into the Z-plane and detection zone was observed with 3mg/mL allowing the least amount of cellular movement. Representative images shown were taken at 2.5x magnification (12 replicates per condition) and are 1519 µm in Z-height)

Cell Migration Assays

Cat.No 1 x 96 well plate

5 x 96 well plates

5 x 384 well plates

OrisTM Pro Cell Migration PROCMA1 Assay - Tissue Culture Treated

PROCMA5

PRO384CMA5

OrisTM Pro Cell Migration Assay - Collagen I Coated

PROCMACC1

PROCMACC5

PRO384CMACC5

Oris™ Cell Migration Assay Tissue Culture Treated

CMA1.101

CMA5.101

OrisTM Cell Migration Assay Collagen I Coated

CMACC1.101

CMACC5.101

OrisTM Cell Migration Assay Fibronectin Coated

CMAFN1.101

CMAFN5.101

OrisTM Cell Migration Assay TriCoated

CMATR1.101

CMATR5.101

Cell Invasion Assays

Cat. No 1 x 96 well plate

3x 96 well plates

Oris™ Pro 96-well Invasion Assay

PROIA1

PROIA3

Oris™ Pro 96-well Invasion Assay Plus

PROIAPLUS1

PROIAPLUS3

41 www.amsbio.com

Other Physiologically Relevant Cell Assays Cultrex® 3-D Proliferation Assays Recent studies indicate that the composition of the extracellular environment influences cellular responses to apoptosis inducing agents implicating a role for extracellular proteins in influencing both toxicity and drug resistance. As a result, this micro-environment must be mimicked during the course of cell-based studies to provide the most accurate translation to animal models. Cultrex® Cell Proliferation Assays were created in an effort to provide more physiologically relevant assessments when using cell models in the screening process for compounds that influence toxicity, cell survival, tumourigenicity, and new tumour formation. These assays offer a flexible, standardized, high-throughput format for quantitating the degree to which pharmacological compounds influence toxicity or tumourigenicity in an in vivo-like environment. The Cultrex® Cell Proliferation Assay has been adapted to multiple formats so that cell proliferation may be evaluated against different extracellular matrices. The assay is available in the following formats: Cat. No

Description

Pack

3445-096-K

Cultrex® 3D Culture BME Cell Proliferation Assay Kit

96 tests

3446-096-K

Cultrex® 3D Culture Laminin I Cell Proliferation Assay Kit

96 tests

3447-096-K

Cultrex® 3D Culture Collagen I Cell Proliferation Assay Kit

96 tests

Percent Proliferation (untreated)

120% 100% 80% 50% 40% 20% 0% Plastic

Collagen

BME

Laminin I

Fig. 45 Proliferation of MDA-MB-231 cells in different extracellular environments in the presence of 50 µl Etoposide. 3D Culture was conducted using the tumorigenicity protocol. Briefly cells were seeded in the presence or absence of ECM proteins and treated after two hours. Cell cultures were incubated at 37◦ C 5% CO2 for 4 days. Then 15 µl of 3D Culture Cell Proliferation Reagent was added to each well, and absorbance at 450 nm was determined at 2 hours. Values were assessed as percentage of untreated controls

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Cultrex® in vitro Angiogenesis Assay Endothelial Cell Invasion Kit During angiogenesis, which is the formation of neo-vasculature associated with wound healing and a host of disease states including tumour growth, endothelial cells invade through the basement membrane to form sprouting vessels, which in turn invade hypoxic tissue. Cultrex® in vitro Angiogenesis Assay Endothelial Cell Invasion Kit was created in an effort to accelerate the screening process for compounds that influence vascular endothelial cell digestion through a basement membrane extract (BME) layer. This kit offers a flexible, standardized, 96 well high-throughput format for quantitating endothelial invasion, in vitro, in response to chemo-attractants and/or inhibiting compounds. This assay employs a simplified Boyden chamber-like design with an 8 micron polyethylene terephthalate (PET) membrane. Sulforaphane [1-isothiocyanato-(4R)-methylsulfinyl)-butane], a naturally occurring cancer chemo-preventive agent, is provided as a control for inhibition of in vitro endothelial cell invasion through Cultrex® BME. Detection of cell invasion is quantified using Calcein-AM. Calcein AM is internalized by the cells, and intracellular esterases cleave the acetomethylester (AM) moiety. Free Calcein fluoresces brightly, and this fluorescence may be used to quantitate the number of cells that have invaded or migrated using a standard curve. Cat. no 3471-096-K

Description Cultrex® In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit

Pack 96 tests

CultreCoat® Vascular Permeability Assays The vascular endothelium is the thin monolayer of cells that lines blood vessels and provides a network for the exchange of biological materials such as gases, nutrients, and metabolic waste throughout the body. The endothelium exhibits a selective barrier function between the vessel lumen and the surrounding tissues which controls this exchange. There are many vasoactive cytokines and growth factors that function in regulating the degree of vascular permeability, such as interleukin 1 alpha and beta, TNF-alpha, IFN gamma, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). There are also several systemic diseases associated with disruption of vascular permeability, such as cancer, diabetes, heart disease, stroke, hypertension, and arthritis.

Fig. 46 Relative Quantification (as a percent of the migration control) of the ability of endothelial cell lines (SVEC4-10, HUVEC and HBMVEC) to travers through 8 micron plyester filters coated with a 1X BME ( Basement Membrane Extract) solution over 24 hour period in response to VEGF, bFGF and other chemoattractants contained in Endothelial Growth Medium-2 (EGM-2), in the presence or absence of 5 µM Sulforaphane. Endothelial Basal Medium (EBM) was used as a negative control due to absence of chemoattractants.

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CultreCoat® Vascular Permeability Assays (Continued) The CultreCoat® Vascular Permeability Assay was created to accelerate the screening process for signal transduction pathway modulators and compounds that influence vascular permeability. This assay offers a flexible, standardized, high-throughput format for quantitating the degree to which genes or compounds can influence the maintenance of endothelial cell-to-cell adhesion. This assay employs a simplified Boyden chamber design with a one micron polyethylene terephthalate (PET) membrane coated with collagen I. Ports within the migration chamber (top) allow access to the assay chamber (bottom) without dismantling the device. This design is easier to use, prevents contamination, and is adaptable for robotic high throughput systems. The assay chamber may be directly analysed in a 96 well plate reader, eliminating transfer steps that introduce additional variability to the assay.

Vascular Permeability Index (5)

Since different cell lines may exhibit variation in size, adhesive properties, and proliferation rates, cell seeding densities and culture periods may require some optimization (the current protocol is optimized using human umbilical vein endothelial cells (HUVECs)). Detection of vascular permeability is quantified using FITC-Dextran. The confluent cell monolayer forms a barrier through cell-cell adhesion mechanisms such as junctions (adherens, tight, and gap) and desmosomes, and this barrier restricts FITC-Dextran to the top chamber. Disruption of these cell-cell adhesion mechanisms creates gaps in the intercellular space that allow diffusion of FITC-Dextran into the bottom chamber, so the amount of FITC-Dextran that diffuses is related to the amount of intercellular space, which is representative of vascular permeability.

Fig. 47 Cytochalasin B increases vascular permeability. HUVECs were seeded at 100,000 cell/well and cultured for 72 hours at 37◦ C in a CO2 incubator. Cells were treated with cytochalasin B for five hours and evaluated for vascular permeability using CultureCoat® 96 Well in vitro Vascualr Permeability Assay.

Cat. no

Description

Pack

3475-024-K

Cultrex® 24 Well InVitro Vascular Permeability Kit

24 tests

3475-096-K

Cultrex® 96 Well InVitro Vascular Permeability Kit

96 tests

44 www.amsbio.com

Cultrex® 3D Culture Cell Harvesting Kit 3D Cultures exhibit cellular behaviors and morphologies similar to those seen in vivo; however, the adaptation of these models for studying biochemical processes has been impeded by the challenge of separating intact cells from extracellular proteins comprising the hydrogel. Commonly, proteases are employed to degrade these extracellular proteins; however, proteases also degrade proteins on the cell surface, and protease activity may carry over into lysate preparations. Non-enzymatic methodologies have also been described for depolymerizing extracellular matrix proteins, although the implementation of these protocols remains problematic for some researchers. Cultrex® 3D Culture Cell Harvesting Kit provides an optimized and standardized solution for the isolation and normalization of cell lysates from 3D Culture Matrix™ BME or Laminin I for subsequent biochemical analysis. Cat. no 3448-020-K

Description

Pack

Cultrex® 3D Culture Cell Harvesting Kit 20 tests

Cultrex® Cell Staining Kit The Cell Staining Kit provides ready-to-use CS Solution and Wash Solution. The CS Solution contains a mixture of Azure A and Methylene Blue specially formulated to provide optimized staining of cells and structures grown on BME. Ideal staining contrast is achieved with minimum background since excess stain can be easily washed away using the wash buffer. The blue coloration allows optimal contrast between cells and the growth matrix. APPLICATIONS: Staining of cells grown on Cultrex® Basement Membrane Extract (BME) or other extracellular membrane protein extracts after applications such as: • • •

Tube formation assay: BME promotes differentiation of endothelial cell lines (e.g. SVEC4-10), into capillary like structures Neurite outgrowth assay: BME promotes differentiation of neurites from chick dorsal root ganglion Aortic ring angiogenesis assay: BME promotes differentiation of rat or mouse aorta tissue to form branched, capillary-like structures that extend radially outward, and into the ring

Cat. no 3437-100-K

Description Cultrex® Cell Staining Kit

Pack 100 ml

Fig. 49 Cell Staining Ki t, Human Umbilical Vein Fig. 49 Cell Staining Kit, Human Umbilical Vein Endothelial Endothelial Cell s (HUVECs) stained with Cultr ex® Cell Cells (HUVECs) stained Cultrex® Cell hours Staining Cells Staining Ki t. Cells werewith gro wn for four onKit. gelled were grown for four hours on gelled Cultrex® BME Reduced Cultrex® BME Reduced Gro wth Factor Growth Factor #3433-005-02) in EGM– 2 medium. (catalog #3 433(catalog -00 5-0 2) in EGM– 2 medium.

Fig. 48 SVEC4-10 cell s sta ined wi th Cultrex® Cell Fig. 48 SVEC4-10 cells stained with Cultrex® Cell Staining Staining Kit. grown Cells on werCultrex® e grown onphenol Cultrex® BME, Kit. Cells were BME, red free in phenolcontaining red free in6% DMEM DMEM FBS containin g 6% FBS

45 www.amsbio.com

TROUBLESHOOTING GUIDE

Alvetex速 Scaffold

Spheroid-based assays

1. PROBLEM: Low signal when reading plate

1. PROBLEM: Spheroids not forming

1. SOLUTION: Perform a growth curve first to determine best cell density. Histology of bigger formats can also be helpful as cell densities can be kept consistent.

1. SOLUTION: Check that cells were centrifuged after mixing with matrix.

2. PROBLEM: Cell attachment is unsatisfactory 2. SOLUTION: Coat the scaffold with an appropriate natural matrix or MAPTrixTM bio-mimetic.

2. PROBLEM: Spheroids not invading Check that invasion matrix was left to gel before final media addition. 2. SOLUTION: Check that invasion matrix was left to gel before final media addition.

Lipidure速-coat plates

Natural Matrices

1. PROBLEM: Spheroids not forming or wrong size

1. PROBLEM: Pre-mature gelling of matrix

1. SOLUTION: Perform a growth curve first to determine best cell number. Add an appropriate natural matrix or MAPTrixTM bio-mimetic to enhance cell attachment and growth.

1. SOLUTION: Make sure that the matrix, as well as all tips and plates are at 4C. Use ice trays as much as you can. 2. PROBLEM: Bubbles appear in matrix 2. SOLUTION: Rigorous pipetting of matrices can lead to warming up and gelling.

MAPtrix速 1. PROBLEM: Low signal when reading plate 1. SOLUTION: Perform a growth curve first to determine best cell number.

OrisTM assays 1. PROBLEM: Low signal when reading plate 1. SOLUTION: Perform a growth curve first to determine best cell density. Consider other read-outs (e.g. proliferation).

46 www.amsbio.com

FIND OUT MORE ABOUT PHYSIOLOGICALLY RELEVANT CELL CULTURE ONLINE

Editor: Dr. Elad Katz Design: Ewa Semenowicz 05/14

www.amsbio.com

CONTACT US: AMS Biotechnology (Europe) Ltd - UK & Rest of World 184 Park Drive, Milton Park, Abingdon OX14 4SE, U.K. T: +44 (0) 1235 828 200 | F: +44 (0) 1235 820 482 AMS Biotechnology (Europe) Ltd â&#x20AC;&#x201C; Switzerland Centro Nord-Sud 2E CH-6934 Bioggio-Lugano. T: +41 (0) 91 604 55 22 | F: +41 (0) 91 605 17 85 AMS Biotechnology (Europe) Ltd - Deutschland T: +49 (0) 69 779099 | F: +49 (0) 69 13376880 amsbio LLC - United States 1035 Cambridge Street, Cambridge, MA 02141 T: +1 (617) 945-5033 or +1.(800) 987-0985 | F: +1 (617) 945-8218

www.amsbio.com | info@amsbio.com

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