Thermo Scientific HyClone HyStem Hydrogels

Transcription

1 Thermo Scientific HyClone HyStem Hydrogels Regulatory Friendly Extracellular Matrix Optimization Peptide and Growth Factor Incorporation 3-D Stem Cell Culture

2 Introduction Introduction Providing optimal culture conditions can be one of the greatest challenges in stem cell research. Aside from optimal nutrient requirements, an optimal substrate is also important. There are several key considerations to take into account when choosing the appropriate substrate on which to expand and differentiate these very important cells. Regulatory friendliness of the substrate Lot-to-lot consistency for reliable results User-controlled rigidity and composition of the substrate for optimal conditions Suitability of the substrate for both in Vitro and in Vivo experimentation Recently, we entered into an agreement with Glycosan Biosystems, Inc., to provide a wide array of extracellular matrices designed specifically to meet the exacting requirements of the stem cell researcher. Sold under the Thermo Scientific brand, the HyStem line of extracellular matrix kits are based on hyaluronic acid (HA) a component of the extracellular matrix (ECM) that is abundant in embryos and in the microenvironments surrounding stem cells. HyStem hydrogels provide a compliant, viscoelastic matrix that is physiologically relevant for stem cells. Choosing a Thermo Scientific HyClone HyStem Whether your requirements mandate an animal component-free system or your research requires the ability to incorporate growth factors and peptides into your stem cell culture system, there is a HyStem Hydrogel kit to meet the needs of your exacting research. Product Characteristics Key Features Key Benefits Applications HyStem HyStem-C HyStem-HP Chemically modified hyaluronan crosslinked with PEGDA HyStem but with chemically modified gelatin added HyStem-C but with chemically modified heparin added 1. Hyaluronan-based 2. Just add water and go 3. Can change formulation or stiffness before gelation 1. Animal-free 2. Simple to use 3. Easy to modify 1. User-designed surface coatings 2. Differentiation of neurospheres in bioreactors 1. Gelatin (or denatured collagen) 1. Starting point for 1. Culture of Neural progenitors, expansion is an excellent additive to start with when optimizing your own matrix/surface optimizing your own surface or matrix. and differentiation of neurons from neurospheres (depending on the ratio of HyStem to Gelin-S) 2. MSC differentiation 1. Contains heparin which acts 1. Slow delivery of 1. Neural stem cell/np cell culture by ionically binding to growth growth factors 2. Culture of neural Schwann cells in vitro factors to cells Table 1. Outlines the characteristics of each of the available Thermo Scientific HyClone HyStem s.

3 Thermo Scientific HyClone HyStem Thermo Scientific HyClone HyStem Animal-free HyStem is a fully-defined matrix allowing researchers to customize a stem cell niche. The key feature of HyStem is complete control over matrix characteristics by changing hydrogel stiffness or adding user-supplied extracellular matrix (ECM) proteins and growth factors. The tailored HyStem can then be used to coat a plate or encapsulate cells. HyStem consists of a hyaluronan-based, synthetic matrix. The material is ready for cell culture in three simple steps reconstitute, mix and use. HyStem will gel at room temperature within 30 minutes with no low temperature or low ph steps in its preparation. If needed, Gelin-S denatured collagen) can be purchased for use as a positive control of cell attachment. HyStem is suitable for in Vitro stem cell culture. HyStem Hydrogel kits are optimal for culturing stem cells whose natural environment is rich in hyaluronic acid (HA). It is xeno-free since its two components are thiol-modified hyaluronan (HyStem) and a thiol-reactive crosslinker, polyethylene glycol diacrylate (Extralink ) 1. Extralink is made by adding acrylate groups to both ends of a polyethylene glycol (PEG) polymer. PEG is derived from petroleum and inorganic sources and contains no animal-sourced materials. The hyaluronic acid used to produce HyStem is made by a proprietary bacterial fermentation process which is 100 percent free of animal-derived raw materials. The resultant materials are pure, low in endotoxin, and widely used in a variety of applications. No animalderived ingredients are used in its production. HyStem can be customized by adding ECM proteins 2 or cell attachment peptides 3 into the hydrogel to provide attachment sites and / or differentiation signals. They can also be varied by changing the hydrogel rigidity 4 to match that of the native cell environment. Applications Stem cell expansion The following stem cells have been cultured in HyStem: Human embryonic stem cells (H9s) 10,11 Umbilical cord blood CD34+ stem cells 12 Hepatic stem cells 13 Hepatic progenitor cells 13 Adipose derived stem cells 14 Mesenchymal stem cells 15,16 Flexibility HyStem Hydrogel gives the researcher complete control over: Hydrogel rigidity Amount and type of ECM protein incorporated Cell attachment peptide incorporation Cell encapsulation or plating on top of hydrogel Cell growth format (96- to 6-well plates and / or tissue culture inserts) Gelation The reconstituted HyStem components are liquids at C. The hydrogel is formed when the crosslinking agent, Extralink, is added to the HyStem liquid. Once the two components are mixed, gelation occurs within 30 minutes. There are no low temperature or low ph steps. The gelation time can be increased by diluting the components with phosphate buffered saline (PBS) or cell culture media. HyStem provides a basic, viscoelastic matrix for stem cell growth. This matrix can be manipulated by the user by changing its composition 2,3 or rigidity 4. Volume and Composition The HyStem comes in two sizes. Ordering Information: Part # Description Kit Size SV SV HyStem HyStem Kit Contains Qty Description Size 2.5 ml 1 HyStem 1 Extralink (polyethylene glycol 7.5 ml 3 HyStem 3 Extralink (polyethylene glycol Note: Since HyStem hydrogels are composed of only HyStem and Extralink, they do not support cell attachment 7. Glycosan BioSystems other hydrogel kits all contain Gelin-S denatured collagen), which promotes cell attachment 7. Since the collagen in Gelin-S is denatured and animal sourced (porcine-derived), it has limited utility for stem cell cultivation where attachment sites can also signal cells to differentiate and where animal sourcing is a concern. The lack of attachment sites is less of a concern for stem cells that are encapsulated 8,9. However, if the researcher plans to plate cells on the surface of the hydrogels, then additional components need to be incorporated into the hydrogel in order to promote cell attachment. ECM proteins can be non-covalently added to HyStem prior to crosslinking or cell attachment peptides can be covalently linked into the matrix. Because the ECM proteins and peptides can also provide differentiation signals as well as attachment signals, the researcher must determine the appropriate type to use. Encapsulated cells are recovered from the HyStem hydrogel by enzyme digestion using hyaluronidase. If cells are plated on the surface, then they are passaged using either trypsin, dispase, collagenase or other gentler detachment solutions such as Thermo Scientific HyQTase.

4 Thermo Scientific HyClone HyStem-C Thermo Scientific HyClone HyStem-C HyStem-C provides an excellent starting point for optimizing the matrix for stem cell culture. Its composition can be easily tailored to find suitable matrix compositions. Unlike an animal-derived ECM, HyStem-C is fully chemically defined. The hydrogels are based on three biocompatible components: thiol-modified hyaluronan (HyStem ), thiolmodified gelatin (Gelin-S), and a thiol-reactive crosslinker, polyethylene glycol diacrylate (Extralink). HyStem-C hydrogels can be customized by adding ECM proteins and by varying the hydrogel compliance to match the stiffness of native tissues. Applications 3-D Stem Cell Culture In addition to stem cell culture on the surface of the hydrogel, HyStem-C provides the basic scaffold for 3-D stem cell growth. Stem cells can be encapsulated during crosslinking 17 where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth 18. Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel or by trypsinization for cells grown on the surface. Gelin-S provides basic cell-attachment sites for cell lines and primary cells 18,19. Several cell types depend on specific ECM components, such as the natural ECM proteins laminin, collagen, fibronectin and vitronectin, to grow and differentiate all of which may be added to the HyStem-C hydrogel. These proteins are easily incorporated noncovalently into the hydrogel prior to gel formation. If Gelin-S will not be used for cell attachment, we recommend HyStem for incorporating ECMs. Flexibility HyStem-C allows customization of experiments: Dimensionality (3-D encapsulation or 2-D plating on top of hydrogel) Cell-growth format (96- to 6-well plates and / or tissueculture inserts) Amount and type of ECM protein incorporated Hydrogel stiffness or rigidity Gelation Reconstituted HyStem-C components remain liquid at 15 to 37 C. The hydrogel is formed when the crosslinking agent, Extralink is added to a mixture of HyStem and Gelin-S. Gelation occurs within 30 minutes after all three components are mixed. No steps depend on low temperatures or low ph. Diluting the components with phosphate buffered saline (PBS) or cell culture medium can increase the gelation time. Volume and Composition The HyStem-C comes in two sizes. Ordering Information: Part # Description Kit Size SV SV HyStem-C HyStem-C Kit Contains Qty Description Size 2.5 ml 1 HyStem 1 Gelin-S gelatin) 1 Extralink 7.5 ml 3 HyStem 3 Gelin-S gelatin) 3 Extralink Recent publications reveal a gel s stiffness is a very potent signal of cell differentiation for MSCs 20. Researchers requiring the ability to significantly control differentiation need to not only optimize the differentiation induction medium, but also the gel stiffness, or rigidity. The rigidity of the HyStem-C hydrogel can be varied either by changing the amount of Extralink used for crosslinking 21 or by diluting the HyStem and Gelin-S solutions using PBS or cell-culture medium. A variety of stem cells (human embryonic, human mesenchymal stem cells 21 ) and progenitor cells (neural progenitor, hepatic progenitor) have been cultured in HyStem-C.

5 Thermo Scientific HyClone HyStem-HP Thermo Scientific HyClone HyStem-HP HyStem-HP is ideal for stem cell applications where slowly released growth factors are crucial in recreating a stem cell microenvironment. HyStem-HP hydrogels contain thiol-modified heparin which allows the slow release of growth factors (GFs) within an easily customizable environment. HyStem-HP is a synthetic ECM that can be injected and crosslinked in situ. Unlike an animal-derived extracellular matrix (ECM), HyStem-HP is fully chemically defined. The HyStem-HP Hydrogel kit contains a combination of thiol-modified hyaluronan, and a thiol-modified heparin (HyStem-HP), thiol-modified gelatin (Gelin-S) and Extralink, a thiolreactive crosslinker. The immobilized heparin in the hydrogel mimics the heparin sulfate proteoglycans normally present in the extracellular matrix (ECM). It also helps protect growth factors (GFs) from proteolysis and slows their release to attached cells 22. This reduces the amount of GF required to achieve stimulation of cell growth or differentiation when compared to the use of free GF in media. All GFs tested to date (bfgf, VEGF, Ang-1, PDGF, TGFβ1, KGF) are released at different rates, but over a period of several weeks Applications 3-D Stem Cell Growth In addition to stem cell culture on the surface of the hydrogel, HyStem-HP provides the basic scaffold for 3-D stem cell growth. Cells can be encapsulated during crosslinking 25, where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth 26. Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel or by trypsinization for cells grown on the surface. Gelin-S provides basic cell-attachment sites for stem cell lines. Several stem cell types depend on specific ECM components to grow and differentiate. To affect specific cell performance, other factors such as growth factors or ECM proteins may be added to the HyStem-HP hydrogel. ECM proteins as well as growth factors are easily incorporated noncovalently into the hydrogel prior to gel formation. 3-D Stem Cell Growth Using GF-Supplemented Media For stem cells cultured with GFs in the media, GFs may be removed from the media and added to the HyStem-HP hydrogel. The hydrogel is used to coat a culture flask and cells are cultured on top of the hydrogel using medium without GFs. Note that GFs are released at different rates. Flexibility HyStem-HP ensures complete control over: Amount and type of growth factors incorporated Cell encapsulation or plating on top of hydrogel Cell-growth format (96- to 6-well plates and / or tissue culture inserts) Amount and type of ECM proteins incorporated Hydrogel stiffness or rigidity Gelation Reconstituted HyStem-HP components remain liquid at 15 to 37 C. The hydrogel is formed when Extralink is mixed with HyStem-HP and Gelin-S. Gelation occurs within 30 minutes after all three components are mixed. No steps depend on low temperature or low ph. Diluting the components with phosphate buffered saline (PBS) or cell culture medium can increase gelation time. Volume and Composition The HyStem-HP comes in two sizes: Ordering Information: Part # Description Kit Size SV SV HyStem-HP HyStem-HP Kit Contains Qty Description Size 2.5 ml 1 HyStem-HP hyaluronic acid with thiol-modified heparin) 1 Gelin-S gelatin) 1 Extralink 7.5 ml 3 HyStem-HP hyaluronic acid with thiol-modified heparin) 3 Gelin-S gelatin) 3 Extralink

6 HyStem References HyStem-C References X.Z. Shu, Y. Liu, F. Palumbo, Y. Luo, G.D. Prestwich, In Situ Crosslinkable Hyaluronan Hydrogels for Tissue Engineering, Biomaterials, 25, (2004). X.Z. Shu, Y. Liu, F. Palumbo, Y, Luo, and G.D. Prestwich, T.D. Mehra, K. Ghosh, X.Z. Shu, G.D. Prestwich, and R.A.F. Clark, Molecular Stenting with a Crosslinked Hyaluronan Derivative Inhibits Collagen Gel Contraction, J. Invest. Dermatol., 126, (2006). * X.Z. Shu, K. Ghosh, Y. Liu, F.S. Palumbo, Y. Luo, R.A. Clark, and G.D. Prestwich, Attachment and Spreading of Fibroblasts on an RGD Peptide-Modified Injectable Hyaluronan Hydrogel, J. Biomed. Mat. Res. 68A, (2004). J. L. Vanderhooft, M. Alcoutlabi, J.J. Magda, and G.D. Prestwich, Rheological Properties of Cross-Linked Hyaluronan-Gelatin Hydrogels for Tissue Engineering, Macromol Biosci., in press (2008). A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification, Cell, 126(4): (2006) J. Vanderhooft and G.D. Prestwich, manuscript in preparation X.Z. Shu, S. Ahmad, Y. Liu, and G.D. Prestwich, Synthesis and Evaluation of Injectable, in situ Crosslinkable Synthetic Extracellular Matrices (secms) for Tissue Engineering, J. Biomed Mater. Res. A, 79A(4), (2006). W.S. Turner, E. Scmelzer, R. McClelland, E. Wauthier, W. Chen, L.M. Reid, Human hepatoblast phenotype maintained by hyaluronan hydrogels, J Biomed Mater Red B Appl Biomater 82(1): (2007). [Note: this material was not provided by Glycosan, but is a precursor technology]. S. Gerecht, J.A. Burdick, L.S. Ferreira, S.A. Twonsend, R. Langer, G. Vunjak- Novakovic, Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells, PNAs 104(27): (2007). [Note: This is a not Glycosan material.] Unpublished data from Xuejun Wen s lab, Clemson University and Medical University of South Carolina. Unpublished data from Liisa Kuhn s lab, University of Connecticut Unpublished date from Linda Kelley s lab, University of Utah, and Glycosan W.S. Turner, L.M Reid, University of North Carolina, manuscript submitted Flynn, G.D. Prestwich, J.L Semple, and K.A. Woodhouse, Adipose Tissue Engineering with Naturally-derived Scaffolds and Adipose-derived Stem Cells, Biomaterials, 28, (2007) Y. Liu, X.Z. Shu, and G.D. Prestwich, Osteochondral Defect Repair with Autologous Bone Marrow-Derived Mesenchymal Stem Cells in an Injectable, in situ Crosslinked Synthetic Extracellular Matrix, Tissue Eng., 12, (2006). Unpublished data from Glycosan G. D. Prestwich, Y. Liu, M. Serban, B. Yu, X. Z. Shu, and A. Scott, 3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery, invited, Adv. Enz. Res., in press (2007). X. Z. Shu, S. Ahmad, Y. Liu, and G. D. Prestwich, Synthesis and Evaluation of Injectable, In Situ Crosslinkable Synthetic Extracellular Matrices (secms) for Tissue Engineering, J. Biomed Mater. Res. A, 79A(4), (2006). X. Z. Shu, Y. Liu, F. Palumbo, G. D. Prestwich, Disulfide-crosslinked Hyaluronan- Gelatin Hydrogel Films: A Covalent Mimic of the Extracellular Matrix for In Vitro Cell Growth, Biomaterials, 24, (2003). A. J. Engler, S. Sen, A. L. Sweeney, D. E. Discher, Matrix elasticity directs stem cell lineage specification, Cell, 126 (4): (2006). Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University, and Medical University of South Carolina. HyStem HP References S. Cai, Y. Liu, X. Z. Shu, G. D. Prestwich, Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor, Biomaterials, 26, (2005). D. B. Pike, S. Cai, K. R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bfgf, Biomaterials, 27, (2006). Unpublished data from G. D. Prestwich, et al, University of Utah, and R. Peattie, et al, University of Oregon. G. D. Prestwich, Y. Liu, M. Serban, B. Yu, X. Z. Shu, and A. Scott, 3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery, invited, Adv. Enz. Res., in press (2007). X. Z. Shu, S. Ahmad, Y. Liu, and G. D. Prestwich, Synthesis and Evaluation of Injectable, In Situ Crosslinkable Synthetic Extracellular Matrices (secms) for Tissue Engineering, J. Biomed Mater. Res. A, 79A(4), (2006). HYSTEM LIMITED USE LICENSE BY EITHER OPENING THIS PACKAGE OR USING THE HYSTEM BASED PRODUCT ENCLOSED (THE PRODUCT ), OR BOTH, YOU AGREE TO THE TERMS OF THIS HYSTEM LIMITED USE LICENSE ( LICENSE ). IF YOU DO NOT SO AGREE, DO NOT EITHER OPEN THIS PACKAGE OR USE THE PRODUCT. FOR A PERIOD OF 30 DAYS FROM THE DATE OF SALE, YOU MAY RETURN THE UNOPENED PRODUCT FOR A FULL REFUND. Glycosan BioSystems Inc. ( Glycosan ) grants to you ( RECIPIENT ) a personal, nontransferable, nonsublicensable, nonexclusive license to use this Product in accordance with the Product Data Sheet included with the Product and/or found on Glycosan s web site ( solely for RECIPIENT s own internal testing purposes in in vitro and/or animal studies and not for any other purpose(s), including but not limited to: (a) administration to humans, for any purpose, in any form whatsoever; or (b) administration to any animal or addition to any food intended for or possibly resulting in human consumption. RECIPIENT shall not use the Product to provide services for any third party on a commercial basis. 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Recipient further agrees to notify Glycosan in writing of any such Invention, and upon request by Glycosan, to enter into good faith negotiations with Glycosan for an exclusive license to any such Invention. THE PRODUCT IS PROVIDED AS IS AND WITH ALL FAULTS, AND TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE LAW, GLYCOSAN HEREBY DISCLAIMS ALL WARRANTIES AND CONDITIONS, WHETHER EXPRESS, IMPLIED OR STATUTORY. If any part of this License is found void or unenforceable, it will not affect the validity of the balance of this License, which shall remain valid and enforceable according to its terms. This Product is sold pursuant to a license from the University of Utah Research Foundation. Patents Pending Thermo Fisher Scientific Inc. All rights reserved. HyStem, HyStem-C and HyStem-HP, DG Water, Extralink, Glycosil and Gelin-S are trademarks of Glycosan Biosystems, Inc, Salt Lake City, Utah. All HyStem kits are manufactured by Glycosan exclusively for Thermo Fisher Scientific. 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