3D Cell Culture Systems

Transcription

1 3D Cell Culture Systems Marshall J. Kosovsky, Ph.D. BD Biosciences February 16, 2011

2 Topics for Discussion Overview of Cell Culture Systems: 2D vs. 3D Extracellular Matrix (ECM) Proteins Epithelial cell differentiation Endothelial cell tube formation Animal-Free Matrix: BD PuraMatrix Peptide Hydrogel Primary hepatocytes differentiation Neuronal cell differentiation Endothelial cell tube formation Cell Culture Insert Platform Tumor cell migration/invasion Endothelial cell migration/invasion General cell differentiation (3D co-culture) 2

3 Cell culture systems: 2D 3D 2D Non-treated (hydrophobic) BD Falcon Tissue Culture (TC)-treated [net (-) charge] BD Primaria [mixture of (-) and (+) charge] BD BioCoat (large variety of ECMs, poly-lysine) BD PureCoat [carboxyl (-), amine (+); animal free] 3D ECM coatings (BD Matrigel matrix, BD Laminin/Entactin, Collagens) Rigid 3D Scaffolds (e.g., PLA, PGA) Animal-free hydrogels (BD PuraMatrix Peptide Hydrogel) Cell culture inserts (e.g., co-culture models such as BBB) Animal models 3

4 2D vs. 3D Cell Culture Growth Substrate 2D Rigid; inert 3D Mimics natural tissue environment Architecture Not physiological; cells partially interact Physiological ; promotes close interactions between cells, ECMs, growth factors Cell Encapsulation No Yes Growth Factor Diffusion Rapid Slow; biochemical gradients regulate cell-cell communication and signaling 4

5 Why Culture Cells in 3D? Tissue and organs exist in 3D Studying cells in 3D enables researchers to mimic or approximate physiological conditions that exist in vivo Strong historical evidence that 3D cell culture works for establishing differentiated cell morphology and function Feature Articles in Nature, August 2003 Goodbye, Flat Biology? "... biologists starting to explore the merits of culturing cells in 3D have been stunned by the difference it makes to the way cells behave, which is much closer to their behavior in vivo." Biology s New Dimension (highlights BD Matrigel matrix and BD PuraMatrix ) In 10 years, anyone trying to use 2D analyses to get relevant and novel biological information will find it difficult to get funded. Mihael Polymeropoulos, CSO, Vanda Pharmaceuticals (former Head of Pharmacogenetics, Novartis) 5

6 Cell Culture Models Support or Promote Cell Behavior Cell behaviors (differentiation, functionality) influenced by cues in the microenvironment: Cell morphology (structure, phenotype) Polarity (functional directionality) Growth (proliferation) Cell motility (migration, invasion) Neurite outgrowth Signal transduction (surface receptor function) Gene and protein expression (different cell types can express different genes/proteins; liver vs. heart vs. brain) Biochemical activities (proteins, enzymes) 6

7 The Extracellular Matrix Complex mixture containing glycoproteins, collagens, and proteoglycans Forms structural framework that stabilizes tissues and provides mechanical support for cell attachment Plays important role in cell functionality and differentiation Receptor-mediated signaling Regulation of gene expression 7

8 The Basal Lamina: A Thin Mat that Underlies Epithelial Cell Sheets and Tubes basal lamina = basement membrane BD Matrigel matrix = reconstituted basement membrane Figure: Molecular Biology of the Cell (3rd Edition) 8

9 Basal Lamina in Chick Embryo Cornea epithelial cells basal lamina collagen fibrils Figure: Molecular Biology of the Cell (3rd Edition) 9

10 ECM Contributes to Intracellular Signaling Pathways ECM-based growth substrates provide a physiological environment that supports and promotes key cell functions ECM molecules interact with cell surface receptors (e.g., regulation of integrin signaling by fibronectin:integrin interactions) ECM appears to function in the storage and presentation of growth factors 10

11 BD Matrigel matrix: Reconstituted Basement Membrane Composition Laminin ~ 60% Collagen IV ~ 30% Entactin ~ 8% Heparan sulfate proteoglycan (perlecan) Growth factors (e.g., PDGF, EGF, TGF-β) Matrix metalloproteinases 11

12 BD Matrigel Matrix High Concentration Protein concentration: ~ mg/ml Applications Human tumor xenograft in immunodeficient nude mice Subcutaneous injection of Matrigel mixture containing human tumor cells (e.g., lung, prostate, breast, colon) BD Matrigel matrix Plug Assay for in vivo angiogenesis Subcutaneous injection of BD Matrigel matrix mixture containing test substances (e.g., antibodies, growth factors, synthetic peptides) and/or tumor cells Removal of plug and analysis of new vessel formation 12

13 High Concentration ECM Proteins for 3D Cell Culture High Concentration Laminin/Entactin Complex Major component of basement membrane in Engelbreth-Holm-Swarm (EHS) mouse tumors Protein concentration: 10 mg/ml Purity >90% by SDS-PAGE Forms 3D gel that models tissue microenvironment in vivo Supports cell differentiation (e.g., mouse submandibular cells, endothelial cell tube formation) High Concentration Collagen I Source: rat tail tendon Protein concentration: 8-11 mg/ml Full-length protein (not treated with pepsin) Purity >90% by SDS-PAGE Forms sturdy gel that provides maximal 3D support matrix 13

14 Factors that Influence Cell Differentiation and Functionality Biological composition of the culture environment (e.g., cell types, ECMs, growth factors) Molecular interactions and cell adhesion (cell:cell, cell:ecm, ECM:ECM, cell:growth factor, and ECM:growth factor) Mechanical strength and structural properties (degree of rigidity, 3D architecture) 14

15 Primary Hepatocytes Exhibit Differentiated Morphology on 3D Growth Substrates Collagen I (2D thin coat) Collagen I (3D gel) BD Matrigel matrix (3D) 15

16 Mammary Epithelial Cell Differentiation in BD Matrigel Matrix using 3D Overlay Method 8 days 20 days Activated caspase 3 Laminin-5 DAPI Phospho-ERM (ezrin/radixin/moesin) hdlg (human disk large) DAPI 15 days Data kindly provided by Dr. Joan Brugge and originally described in Debnath J, et al. (2003) Methods 30:

17 Human Microvascular Endothelial Cells Form Tubules on BD Matrigel Matrix Collagen I 2D BD Matrigel matrix 3D Bright Field BD Matrigel matrix 3D 17

18 Endothelial Cell Tubulogenesis: High Resolution Detection Using Confocal Collapsed Stack Imaging Non-confocal single plane Confocal single plane Confocal collapsed stack BD HUVEC-2 cells, stained with Calcein AM, 4X confocal images on BD Pathway Bioimager 18

19 Concentration-Dependent Inhibition of Endothelial Cell Tube Formation by Suramin Calcein AM staining, 4x confocal µm 19

20 Different Analysis Parameters - Similar Results Total Tube Length (pixels) IC 50 = 2.93E Log [M] Suramin Total Tube Area (pixels) Tube Complexity (total # of segments) Log [M] Suramin IC 50 = 2.94E-05 IC 50 = 2.97E Log [M] Suramin

21 Potential Limitations of Current Systems Composition of the environment not optimal for key cell type(s) Growth substrate or material not well defined Complex materials often exhibit lot-to-lot variability Presence of animal-derived components Synthetic materials may generate acidic breakdown products that are cytotoxic 21

22 BD PuraMatrix Peptide Hydrogel A Synthetic Biomaterial for Optimizing 3D Cell Culture Environments Developed at Massachusetts Institute of Technology, Cambridge, MA Composed of synthetic peptide (1% w/v) and 99% water Salt-mediated assembly into 3D hydrogel with average pore size of nm Supports attachment & differentiation of cell types such as primary hepatocytes, neurons, endothelial cells, chondrocytes, neural & mesenchymal stem cells Suitable for in vivo studies of tissue regeneration and repair Biocompatible, devoid of animal-derived materials & pathogens PuraMatrix is a trademark of 3DM, Inc., Cambridge, MA 22

23 Synthetic Peptide Composition 16 amino acid sequence; 1% peptide solution (w/v) Alternating hydrophilic and hydrophobic side chains Promotes cell attachment, but does not require integrin-based adhesion Images of molecular models kindly provided by Dr. Shuguang Zhang, Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA 23

24 Salt-Mediated Molecular Self-Assembly into Nanofiber Structure single peptide nanofibers Ionic and hydrogen bonding Hydrophobic and van der Waals interactions Nanofibers exhibit β-sheet bilayer structure 24

25 Nanofiber β-sheets Further Organize into Macroscopic Scaffolds nanofiber scaffold AFM image provided by Dr. Shuguang Zhang, MIT 25

26 SEM Images of Macroscopic Structure Images of BD PuraMatrix Peptide Hydrogel at increasing magnification At lower magnification, the scaffold exhibits felt-like appearance (a). Higher magnification reveals interwoven peptide fibers approximately nm in diameter (f). Originally described in Holmes, T.C., et al. (2000) PNAS, Vol 97, pp

27 SEM Analysis of Macroscopic Structure: BD PuraMatrix vs. BD Matrigel Matrix BD Matrigel matrix BD PuraMatrix Data kindly provided by Dr. Shuguang Zhang and originally described in Gelain, F, et al. (2006) PLoS ONE 1(1):e

28 The Assembled Hydrogel is Comprised of 99% water Hydrated hydrogel environment promotes effective diffusion of nutrients and macromolecules Samples readily visualized using standard staining and microscopy Images provided by Dr. Shuguang Zhang, MIT 28

29 Scanning Electron Microscopy of Rat Primary Hepatocyte Spheroids on BD PuraMatrix Peptide Hydrogel Spheroids attached on the surface and embedded within the hydrogel (white arrows) High magnification view of a spheriod Data kindly provided by Sihong Wang and originally described in Wang, S, et al. (2008) Tissue Engineering Part A 14(2):

30 BD PuraMatrix Peptide Hydrogel: Neuronal Cell Differentiation Rat PC12 cell neurite outgrowth. The image is a merged stack of multiple confocal optical sections. Primary rat hippocampal neurons form active synapses. Confocal image of synaptically active neuronal membranes. Data provided by 3DM, Inc. and originally described in Holmes, TC, et al. (2000) PNAS USA 97:

31 BD PuraMatrix Peptide Hydrogel: Human Umbilical Vein Endothelial Cells A B HUVECs encapsulated within BD PuraMatrix elongate and form interconnected microvascular networks Cells cultured for 2 days in endothelial cell growth medium supplemented with 50 ng/ml VEGF and 50 ng/ml PMA. (A) 0.74 mg/ml BD PuraMatrix; (B) 1.3 mg/ml BD PuraMatrix Data kindly provided by Dr. Alisha Sieminski 31

32 Representative References BD PuraMatrix Peptide Hydrogel Cell/Tissue Type Bone Cartilage Hepatocyte Endothelial Neuronal Reference Misawa, H., et al. (2006) PuraMatrix facilitates bone regeneration in bone defects of calvaria in mice. Cell Transplant. 15(10):903. Yamaoka, H., et al. (2006) Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials. J. Biomed. Mater Res A. 78(1):1. Wang, S., et al. (2008) Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel. Tissue Engineering Part A 14(2):227. Sieminski, AL, et al. (2008) Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J Biomed Mater Res A 87:494. Thonhoff, J.R., et al. (2008) Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res. 1187:42. 32

33 Cell Culture Inserts: Membrane Supports for Complex Cellular Assays Cell culture inserts provide a two compartment culture system suitable for a variety of complex cellular assays Insert wells contain a microporous membrane floor composed of polyethylene terephthalate (PET) available with different pore diameters Pores allow exchange of media, nutrients, molecules and the passage of cells (pore size dependent) Small pore diameters (0.4 and 1.0 µm) prevent cell passage Large pore diameters (3.0 and 8.0 µm) allow cell passage 33

34 Cell Culture Inserts: BD FluoroBlok Platform A unique fluorescence-blocking membrane ideal for analysis of cell migration and invasion Angiogenesis Endothelial Cell Migration/Invasion Tumor Cell Biology Tumor Cell Migration/Invasion 3D models for metastasis Inflammation Monocyte, Leukocyte Chemotaxis Transendothelial Cell Migration Drug Discovery (single parameter or multiplexing) 34

35 BD FluoroBlok PET Membrane Treated with proprietary blue dye Unique fluorescence blocking membrane blocks light transmission from nm Quantitative analysis using fluorescence detection Increases productivity and assay throughput No need to dismantle, wash and manually count cells Eliminates multiple handling steps: just add cells, label and read 8 µm pores visible light 35

36 BD Falcon FluoroBlok Cell Culture Inserts The blue dyed membrane physically and visually separates cells above the membrane from those below the membrane. Insert (individual or multiwell) Apical Chamber Cross section (not to scale) Track-Etched Pores Basal Chamber Base plate 3 and 8 µm pore diameters 24-well Individual, 24-Multiwell, and 96-Multiwell formats 36

37 BD FluoroBlok Assays are Analyzed using Fluorescent Dyes Appropriate fluorescent dyes exhibit emission wavelength between nm [e.g., BD Calcein AM (green) and BD DiIC 12 (3) (red)] Cell Labeling Methods Pre-Labeling (label cells prior to the assay) Post-Labeling (label cells on underside of membrane after assay) Transfected cells (e.g, transient or permanent expression of GFP) See BD Biosciences Technical Bulletin #451 - Compatible Fluorophores and Dyes for BD Falcon FluoroBlok Inserts and Insert Systems 37

38 The Angiogenesis Pathway Pseudopodial Endothelial Vacuole Vacuoles join to extension cell division formation create lumen Release of pro-angiogenic factors Receptor activation MMP release Cell migration, invasion, and proliferation Figure: Adapted from Molecular Biology of the Cell (3rd Edition) 38

39 BD BioCoat Angiogenesis Systems Endothelial Cell Migration 24- or 96-Multiwell FluoroBlok Insert (3 µm pore size) Coated with Human Fibronectin Endothelial Cell Invasion 24-Multiwell FluoroBlok Insert (3 µm pore size) Coated with BD Matrigel matrix Endothelial Cell Tube Formation 96-well black/clear microplate coated with BD Matrigel matrix (non-insert system) BD Human Umbilical Vein Endothelial Cells (BD HUVEC-2) Pre-qualified for VEGF responsiveness and for use with cell migration assay; also suitable for cell invasion and tube formation assays 39

40 Analysis of Endothelial Cell Migration and Invasion Using BD FluoroBlok Membrane Inserts Fibronectin (migration) or BD Matrigel matrix (invasion) Attractant Excitation (485 nm) Emission (530 nm) BD FluoroBlok PET Membrane (3 μm pores) 40

41 Endothelial Cells Exhibit Concentration- Dependent Migration Towards VEGF BD HUVEC-2 Cells 12 HAEC Cells mean + se (n=4) Fold increase over control mean + se (n=4) VEGF (ng/ml) 3-4 fold stimulation 8-10 fold stimulation Cell migration assessed using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (Fibronectin-coated BD FluoroBlok ; 96-well format). 41

42 BD BioCoat Tumor Invasion Systems Combined Benefits of BD FluoroBlok and BD Matrigel matrix Optimized Protocols 24- and 96-well Formats (8.0 µm pores) 42

43 Analysis of Tumor Cell Invasion Using BD FluoroBlok Membrane Inserts BD Matrigel matrix (invasion) Attractant Excitation (485 nm) Emission (530 nm) BD FluoroBlok PET Membrane (8 µm pores) 43

44 MDA-MB-231 Human Breast Adenocarcinoma Cell Invasion Through BD Matrigel Matrix No need to disrupt sample for manual counting Automated quantitation using bottom-reading fluorescence plate reader Fluorescently labeled cells on underside of BD FluoroBlok membrane post-invasion Option to perform real-time kinetic analysis 44

45 Inhibition of MDA-MB-231 Cell Invasion Through BD Matrigel Matrix by Doxycycline 45

46 Use of Cell Culture Inserts to Establish a 3D Co-Culture Model Cell type Y seeded on underside of membrane (e.g., BBB); cells may physically interact through pores Cell type X seeded on top of membrane Top of insert membrane may be coated with 2D or 3D matrix Clear or BD FluoroBlok PET Membrane Cell type Z seeded on floor of well, physically separated from other cell type(s); potential for paracrine regulation Well floor may be coated with 2D or 3D matrix 46

47 Representative Cell Culture Insert References Cell Type Endothelial Endothelial Monocytes Pancreatic cancer cells PC3 prostate carcinoma Surface Fibronectin (BD BioCoat Angio Migration System) Gelatin-coated BD FluoroBlok (migration) Col I-coated BD FluoroBlok (migration) BD Matrigel matrix (BD BioCoat Tumor Invasion System) BD Matrigel matrix (BD BioCoat Tumor Invasion System) Reference Daneman, R, et al. (2009) PNAS, Vol. 106, pp Cote, MC, et al. (2010) Journal of Biological Chemistry, Vol. 285, pp Ramirez, SH, et al. (2008) Journal of Immunology, Vol. 180, pp Li, Y, et al. (2010) Cancer Research, Vol. 70, pp Kong, D, et al. (2008) Cancer Research, Vol. 68, pp

48 Questions? Contact information: Marshall Kosovsky, PhD Technical Support: tel: bdbiosciences.com/webinars For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are property of Becton, Dickinson and Company BD 48