Introduction
For decades, researchers have cultured mammalian cells on tissue culture plastic not only for basic research but also for applied drug discovery and development activities. These two-dimensional surfaces poorly replicate the three-dimensional microenvironment that a cell normally interacts with, inhibiting the cell’s ability to manifest its native phenotype. For example, tumorigenic immortalized mammary epithelial cells (MECs) are poorly distinguishable from its non-tumorigenic counterparts when both are cultured on 2-D tissue culture plastic (Petersen et al 1992). In contrast, within 10 days of encapsulated growth in rodent EHS tumor basement membrane extract (BME), significant differences occur. The non-tumorigenic MECs form organized, polarized acini that eventually reach a normal quiescent state. These changes are comparable to those seen with normal freshly isolated MECs (Weaver et al 1997). In contrast, tumorigenic MECs are solid, disorganized, and continue to divide uncontrollably (Petersen et al 1992). The reason for this difference is due to the overexpression of the tumorigenic cells’ b1 integrin receptor whose effects only manifest themselves in a laminin-rich 3-D culture (Weaver et al 1997).
Three-dimensional cell culture has become increasing important for culturing and differentiating adult, embryonic, and induced pluripotent stem cells because eventual human cell therapeutic applications depend upon their successful culture both in vitro and in vivo. In vitro optimization of stem cell culture requires the adjustment of a variety of extracellular signals including the collection of both insoluble attachment factors and soluble growth factors (Flaim et al 2006; Flaim et al 2008; Soen et al 2006) and matrix stiffness (Engler et al 2006). The resultant vector of their effects points the undetermined cell along a developmental lineage path that is crucial to stay on before cellular transplantation.
In contrast, less attention has been placed on in vivo cell culture (transplantation). This second step in translational process also requires 3-D cell culture to provide a suitable physiological milieu for therapeutic cells once they are transplanted. To be clear, transplantation of naked cells devoid of a suitable matrix is akin to 0-D cell culture: the cells have no attachment sites in their vicinity, resulting in anoikis (apoptosis) (Zvibel et al 2002). In addition, the cells will rapidly diffuse away from the point of injection (Mooney and Vandenburgh, 2004) and will likely be unprotected amongst the host’s inhospitable immune response. In sum, cell therapy becomes highly inefficient with only a small percentage of cells engrafting to the host tissues, suggesting massive cell death occurs shortly after transplantation (LaFlamme et al 2007; Terrovitis et al 2008).
Efficient cell therapy then depends on using a suitable injectable matrix that can not only be easily customized in its composition and stiffness but that can also provide a physiological environment which provides attachment sites, localization, and protection from the host immune system. Additionally, the matrix must be FDA approvable and consistent from lot-to-lot. Mouse EHS BME is perhaps the most popular matrix used for cell culture but its rodent origin introduces a variety of safety and consistency concerns for human cell therapies. Self-assembling peptides and collagen I matrices are also widely used but are at pH 2-3 before cell introduction, providing for a harsh cellular environment. Herein we describe a novel hyaluronate-based hydrogel called HyStem which fulfills both the in vitro and in vivo criteria listed above.
HyStem Technology
HyStem hydrogels are based on thiol-modified hyaluronic acid (HA) which endows biocompatibility, physiological relevance, and customizability. HA is the simplest glycosaminoglycan and is a major constituent of the extracellular matrix. HyStem utilizes polyethylene glycol diacrylate (PEGDA) as a crosslinker to convert thiol-modified HA from a liquid solution to a hydrogel in approximately 20 minutes. This reaction can take place at either at physiologic conditions i.e. pH 7.4 and 37 ºC or at room temperature. Additionally, HyStem hydrogels can be prepared in combination with thiol-modified gelatin (Gelin-S) to provide cell attachment sites.
Since gelation occurs in 20 minutes, HyStem allows the user to customize by adding various components such as proteins, growth factors or cells directly into the hydrogel before gelation. Additionally, the crosslinker concentration and composition can be varied to alter hydrogel properties since the local matrix stiffness has important implications for cell development and differentiation (Engler et al 2006). To this end, the stiffness of HyStem hydrogels can be varied between 15 and 3500 Pa simply by changing the initial thiol-modified HA and gelatin concentrations as well as the crosslinker concentration (Vanderhooft et al 2009). Further customization can be performed with the HyStem-HP hydrogel kits. These kits contain an addition component, thiol-modified heparin, to help regulate the control of growth factors from the hydrogel. Small amounts of the modified heparin can slow the release of cytokines and growth factors in vitro and in vivo (Peattie et al 2008). Release profiles for six different growth factors were unique to each factor and found to exhibit first order exponential kinetics. Additionally, hydrogels containing vascular endothelial growth factor or angiopoietin-1 produced twice the vascularization response indicating that incorporated growth factors retain their physiologic effectiveness.
An important feature for hydrogels used for 3-D cell culture is the ability to gently and rapidly recover encapsulated cells from the hydrogel. Commercially available matrices usually require either mechanical disruption (self-assembling peptides), incubation on ice (mouse basement membrane extract) or proteinases such as collagenase (Collagen I). These types of manipulations can adversely affect cell viability and its gene and protein expression profiles. In contrast, HyStem allows the gentle recovery of encapsulated cells when HyStem is prepared with a new modified crosslinker which allows rapid dissolution under mild reducing conditions. Disulfide moieties were incorporated into the center of polyethylene glycol diacrylate molecules (Zhang et al 2009). The resulting crosslinkers (called PEGSSDA) in combination with HyStem produce hydrogels that dissociate in the presence of N-acetyl cysteine in about one hour. Following expansion, the encapsulated cells were recovered with both a high yield and viability (Zhang et al 2009).
In Vitro and In Vivo Examples
HyStem lends itself to investigate basic matrix biology since its composition and stiffness can be easily altered. 3-D culture in the presence of thiol-modified HA combined with Gelin-S, collagen I, or laminin significantly changes both the metabolic profiles and proliferative state of hepatic stem cells and hepatoblasts (Turner et al 2008). HyStem hydrogels have also been used in culturing human embryonic stem cells (hESCs) to investigate changes in vimentin synthesis when moving hESCs from feeder layers to feeder- free conditions. An increase in vimentin levels when moving to feeder-free conditions is hypothesized to correlate with the transition from an epithelial to a mesenchymal-type phenotype (Ulmann et al, 2007). By growing hESCs on a softer HA-based hydrogel a reduction of vimentin synthesis occurs; this result indicates that vimentin levels could relate to a substrate stress response rather than differentiation (Van Hoof et al 2008).
By virtue of its flexibility as well as its injectability before gelation, HyStem provides a bridge from in vitro experiments to in vivo implantation. In particular, HyStem provides not only attachment sites for the incorporated cells but also localization. One such example is the delivery of mesenchymal stem cells (MSCs) using thiol-modified HA hydrogels as a synthetic extracellular matrix (sECM). A defect was created in the femoral articular cartilage followed by transplantation of MSCs alone or MSCs encapsulated in hydrogel. At twelve weeks post treatment the MSCs + sECM groups’ defects were completely filled with firm, elastic, translucent cartilage and showed superior integration of the repair tissue with the normal cartilage compared to MSCs alone or untreated controls (Liu et al 2006).
Another application of MSCs is as a cell therapy vehicle. Since MSCs may also play a role in tumor growth and metastasis it is undesirable to inject them systematically. Thiol-modified HA hydrogels can localize and support encapsulated MSCs that were engineered to express a therapeutic recombinant antibody. These cells were mixed with the hydrogel and injected into a location distant from tumors developing in mice. Experimental groups exhibited an effective anti-tumor response and tumor regression while maintaining localization of the injected MSCs. This type of therapy is particularly appealing as the localized cells can be removed following the desired effect.
Summary
Due to the unique yet simple chemistry, HyStem is ideally suited for researchers interested in customized 3-D cell culture and translation therapies. The physiological milieu provided for cells allows researchers to optimize and improve cell response and survival. Additionally, cell response to various environments can be easily investigated and tuned to produce specific outcomes. With Cell Therapy’s requirement for homogeneity and consistency in its pluripotent cell populations and differentiated offspring (Carpenter et al, 2009), HyStem’s tunability may very well become its most important attribute.
References
Carpenter MK, Frey-Vasconcells J, and Rao MS, Developing safe therapies from human pluripotent stem cells, Nat. Biotech 27(1): 607-613, 2009.
Compte M, Cuesta AM, Sanchez-Martin D, Alonso Camino V, Vicario JL, Sanz L, Alvarez-Vallina L.Tumor Immunotherapy Using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds. Stem Cells, 27, 753–760, 2009.
Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006 Aug 25;126(4):677-89.
Flaim CJ, Chien S, Bhatia SN. An extracellular matrix microarray for probing cellular differentiation. Nat Methods. 2005 Feb;2(2):119-25.
Flaim CJ, Teng D, Chien S, Bhatia SN. Combinatorial signaling microenvironments for studying stem cell fate. Stem Cells Dev. 2008 Feb;17(1):29-39.
Laflamme MA, et al., Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007 Sep;25(9):1015-24.
Liu Y, Shu XZ, Prestwich GD, Osteochondral Defect Repair with Autologous Bone Marrow-Derived Mesenchymal Stem Cells in an Injectable, In Situ Crosslinked Synthetic Extracellular Matrix. Tissue Engineering 12(12) 12, 3405-3416, 2006.
Liu Y, Shu XZ, Prestwich GD, Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Cross-Linked Hyaluronan-Derived Hydrogels. Tissue Engineering 13(5), 3405-3416, 2007.
Mooney DJ, Vandenburgh H. Cell delivery mechanisms for tissue repair. Cell Stem Cell. 2008 Mar 6;2(3):205-13.
Peattie RA et al., Effect of gelatin on heparin regulation of cytokine release from hyaluronan-based hydrogels. Drug Deliv. 2008 Aug;15(6):389-97.
Petersen OW, Ronnov-Jessen L, Howlett AR, and Bissell MJ, Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells, Proc. Natl. Acad. Sci. USA 89:9064-9068 (1992).
Soen Y, Mori A, Palmer TD, Brown
Terrovitis J et al, Ectopic Expression of the Sodium-Iodide Symporter Enables imaging of Transplanted cardiac stem cells in vivo by single-photon emission computed tomography or positron emission tomography. Journal of the
Turner WS et al, Nuclear magnetic resonance metabolomic footprinting of human hepatic stem cells and hepatoblasts cultured in hyaluronan-matrix hydrogels. Stem Cells. 2008 Jun;26(6):1547-55.
Ullmann U, Veld PI, Gilles C, Sermon K, De Rycke M, Van de Velde H, Van Steirteghem A, Liebaers I. Epithelial–mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Molecular Human Reproduction 13(1), 21-32, 2007.
Vanderhooft JL, Alcoutlabi M, Magda JJ, Prestwich GD. Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering. Macromol Biosci. 2009 Jan 9;9(1):20-8.
Van Hoof D, Braam SR, Dormeyer W, Ward-Van Oostwaard D, Heck AJR, Krijgsveld J, Mummery CL. Feeder-Free Monolayer Cultures of Human Embryonic Stem Cells. Express an Epithelial Plasma Membrane Protein Profile. Stem Cells, 26, 2777-2781, 2008.
Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, and Bissell MJ, Reversion of the Malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies, J. Cell Biol. 137 (1): 231-245 (1997).
Zhang J, Skardal A, Prestwich GD, Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery. Biomaterials 29:4521-31, 2008.
Zvibel I, Smets F, Soriano H. Anoikis: roadblock to cell transplantation? Cell Transplant. 2002;11(7):621-30.