The thermoresponsive behavior of a Methylcellulose (MC) polymer was systematically investigated to determine its usability in constructing MC based hydrogel systems in cell sheet engineering applications. Solution-gel analyses were made to study the effects of polymer concentration, molecular weight and dissolved salts on the gelation of three commercially available MCs using differential scanning calorimeter and rheology. For investigation of the hydrogel stability and fluid uptake capacity, swelling and degradation experiments were performed with the hydrogel system exposed to cell culture solutions at incubation temperature for several days. From these experiments, the optimal composition of MC-water-salt that was able to produce stable hydrogels at or above 32 °C, was found to be 12% to 16% of MC (Mol. wt. of 15,000) in water with 0.5× PBS (~150mOsm). This stable hydrogel system was then evaluated for a week for its efficacy to support the adhesion and growth of specific cells in culture; in our case the stromal/stem cells derived from human adipose tissue derived stem cells (ASCs). The results indicated that the addition (evenly spread) of ~200 μL of 2 mg/mL bovine collagen type -I (pH adjusted to 7.5) over the MC hydrogel surface at 37 °C is required to improve the ASC adhesion and proliferation. Upon con？uence, a continuous monolayer ASC sheet was formed on the surface of the hydrogel system and an intact cell sheet with preserved cell–cell and cell–extracellular matrix was spontaneously and gradually detached when the grown cell sheet was removed from the incubator and exposed to room temperature (~30 °C) within minutes.
References
[1]
Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials 2005, 26, 6415–6422, doi:10.1016/j.biomaterials.2005.04.061.
[2]
Muschler, G.F.; Nakamoto, C.; Griffith, L.G. Engineering principles of clinical cell-based tissue engineering. J. Bone Joint Surg. Am. 2004, 86, 1541–1558.
[3]
Sittinger, M.; Bujia, J.; Rotter, N.; Reitzel, D.; Minuth, W.W.; Burmester, G.R. Tissue engineering and autologous transplant formation: Practical approaches with resorbable biomaterials and new cell culture techniques. Biomaterials 1996, 17, 237–242, doi:10.1016/0142-9612(96)85561-X.
[4]
Hubbell, J.A.; Massia, S.P.; Desai, N.P.; Drumheller, P.D. Endothelial cell-selective materials for tissue engineering in the vascular graft via a new receptor. Biotechnology 1991, 9, 568–572, doi:10.1038/nbt0691-568.
[5]
Griffith, L.G.; Naughton, G. Tissue engineering: Current challenges and expanding opportunities. Science 2002, 295, 1009–1014, doi:10.1126/science.1069210.
[6]
Griffith, L.G. Emerging design principles in biomaterials and scaffolds for tissue engineering. Ann. N. Y. Acad. Sci. 2002, 961, 83–95, doi:10.1111/j.1749-6632.2002.tb03056.x.
[7]
Bauer, T.W.; Muschler, G.F. Bone graft materials. An overview of the basic science. Clin. Orthop. Relat. Res. 2000, 371, 10–27, doi:10.1097/00003086-200002000-00003.
[8]
Sikavitsas, V.I.; Bancroft, G.N.; Mikos, A.G. Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor. J. Biomed. Mater. Res. 2002, 62, 136–148, doi:10.1002/jbm.10150.
[9]
Kumar, P.R.A.; Varma, H.K.; Kumary, T.V. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering. Biomed. Mater. 2007, 2, 48–54, doi:10.1088/1748-6041/2/1/008.
[10]
Kumar, P.R.A.; Sreenivasan, K.; Kumary, T.V. Alternate method for grafting thermoresponsive polymer for transferring in vitro cell sheet structures. J. Appl. Polym. Sci. 2007, 105, 2245–2251, doi:10.1002/app.26221.
[11]
Avgoustiniatos, E.S.; Colton, C.K. Effect of external oxygen mass transfer resistances on viability of immunoisolated tissue. Ann. N. Y. Acad. Sci. 1997, 831, 145–167, doi:10.1111/j.1749-6632.1997.tb52192.x.
[12]
Hutmacher, D.W. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000, 21, 2529–2543, doi:10.1016/S0142-9612(00)00121-6.
[13]
Sistino, J.J. Bioreactors for tissue engineering—A new role for perfusionists? J. Extra Corpor. Technol. 2003, 35, 200–202.
[14]
Martin, I.; Wendt, D.; Heberer, M. The role of bioreactors in tissue engineering. Trends Biotechnol. 2004, 22, 80–86, doi:10.1016/j.tibtech.2003.12.001.
[15]
Janssen, F.W.; Oostra, J.; Oorschot, A.; van Blitteswijk, C.A. A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: In vivo bone formation showing proof of concept. Biomaterials 2006, 27, 315–323, doi:10.1016/j.biomaterials.2005.07.044.
[16]
Dvir, T.; Benishti, N.; Shachar, M.; Sohen, S. Novel perfusion bioreactor providing a homogenous milieu for tissue regeneration. Tissue Eng. 2006, 12, 2843–2852, doi:10.1089/ten.2006.12.2843.
[17]
Khong, Y.M.; Zhang, J.; Zhou, S.; Cheung, C.; Doberstein, K.; Samper, V.; Yu, H. Novel intra-tissue perfusion system for culturing thick liver tissue. Tissue Eng. 2007, 13, 2345–2356, doi:10.1089/ten.2007.0040.
[18]
Yamato, M.; Utsumi, M.; Kushida, A.; Konno, C.; Kikuchi, A.; Okano, T. Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng. 2001, 7, 473–480, doi:10.1089/10763270152436517.
[19]
Yamao, M. Cell sheet engineering: from temperature-responsive culture surfaces to clinics. In European Cells and Materials: Biosurf. V - Functional Polymeric Surfaces in Biotechnology; ETH: Zürich, Switzerland, 2003; pp. 26–27.
[20]
Shimizu, T.; Sekine, H.; Yang, J.; Isoi, Y.; Yamato, M.; Kikuchi, A.; Kobayashi, E.; Okano, T. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, Vascularized myocardial tissues. FASEB J. 2006, 20, 708–710.
[21]
Hayashida, Y.; Nishida, K.; Yamato, M.; Yang, J.; Sugiyama, H.; Watanabe, K.; Hori, Y.; Maeda, N.; Kukuchi, A.; Okano, T.; et al. Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze. Invest. Ophthalmol. Vis. Sci. 2006, 47, 552–557, doi:10.1167/iovs.05-0995.
[22]
Akiyama, Y.; Kushida, A.; Yamato, M.; Kikuchi, A.; Okano, T. Surface characterization of Poly(N-Isopropylacrylamide) grafted tissue culture polystyrene by electron beam irradiation, Using atomic force microscopy, And X-ray photoelectron spectroscopy. J. Nanosci. Nanotechnol. 2007, 7, 796–802, doi:10.1166/jnn.2007.509.
[23]
Matsuda, N.; Shimizu, T.; Yamato, M.; Okano, T. Tissue engineering based on cell sheet technology. Adv. Mater. 2007, 19, 3089–3099, doi:10.1002/adma.200701978.
[24]
Chen, C.H.; Tsai, C.C.; Chen, W.; Mi, F.L.; Liang, H.F.; Chen, S.C.; Sung, H.W. Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels. Biomacromolecules 2006, 7, 736–743, doi:10.1021/bm0506400.
[25]
Li, L.; Shan, H.; Yue, C.Y.; Lam, Y.C.; Tam, K.C.; Hu, X. Thermally induced association and dissociation of methylcellulose in aqueous solutions. Langmuir 2002, 18, 7291–7298, doi:10.1021/la020029b.
[26]
Zheng, P.; Li, L.; Hu, X.; Zhao, X. Sol-gel transition of methylcellulose in phosphate buffer saline solutions. J. Polym. Sci., Part. B: Polym. Phys. 2004, 42, 1849–1860, doi:10.1002/polb.20070.
[27]
Tate, M.C.; Shear, D.A.; Hoffman, S.W.; Stein, D.G.; LaPlaca, M.C. Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury. Biomaterials 2001, 22, 1113–1123, doi:10.1016/S0142-9612(00)00348-3.
[28]
Zuk, P.A.; Zhu, M.; Mizuno, H.; Huang, J.; Futrell, J.W.; Katz, A.J.; Benjaim, P.; Lorenz, H.P.; Hedrick, M.H. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng. 2001, 7, 211–228, doi:10.1089/107632701300062859.
[29]
Gimble, J.; Guilak, F. Adipose-derived adult stem cells: Isolation, Characterization, and differentiation potential. Cytotherapy 2003, 5, 362–369, doi:10.1080/14653240310003026.
[30]
Thirumala, S.; Gimble, J.M.; Devireddy, R.V. Transport phenomena during freezing of adipose tissue derived adult stem cells. Biotechnol. Bioeng. 2005, 92, 372–383, doi:10.1002/bit.20615.
[31]
Thirumala, S.; Zvonic, S.; Floyd, E.; Gimble, J.M.; Devireddy, R.V. The effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells. Biotech. Prog. 2005, 21, 1511–1524, doi:10.1021/bp050007q.
[32]
Mitchell, J.B.; McIntosh, K.; Zvonic, S.; Garrett, S.; Floyd, Z.E.; Kloster, A.; Di Halvorsen, Y.; Storms, R.W.; Goh, B.; Kilroy, G.; et al. Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. Stem Cells 2006, 24, 376–385, doi:10.1634/stemcells.2005-0234.
Ford, J.L. Thermal analysis of hydroxypropylmethylcellulose and methylcellulose: Powders, Gels and matrix tablets. Int. J. Pharm. 1999, 179, 209–228, doi:10.1016/S0378-5173(98)00339-1.
[35]
Sarkar, N. Thermal gelation properties of methyl and hydroxypropyl methylcellulose. J. Appl. Polym. Sci. 1979, 24, 1073–1087, doi:10.1002/app.1979.070240420.
[36]
Sarkar, N.; Walker, L.C. Hydration-dehydration properties of methylcellulose and hydroxypropylmethylcellulose. Carbohydr. Polym. 1995, 27, 177–185, doi:10.1016/0144-8617(95)00061-B.
[37]
Heymann, E. Studies on sol-gel transformations. I. The inverse sol-gel transformation of methylcellulose in water. Trans. Faraday Soc. 1935, 31, 846–864, doi:10.1039/tf9353100846.
[38]
Wang, Q.; Li, L. Effects of molecular weight on thermoreversible gelation and gel elasticity of methylcellulose in aqueous solution. Carbohydr. Polym. 2005, 62, 232–238, doi:10.1016/j.carbpol.2005.07.030.
[39]
Xu, Y.; Wang, C.; Tam, K.C.; Li, L. Salt-assisted and salt-suppressed sol-gel transitions of methylcellulose in water. Langmuir 2004, 20, 646–652, doi:10.1021/la0356295.
[40]
Procter, H.R. The equilibrium of dilute hydrochloric acid and gelatin. J. Chem. Soc. Trans. 1914, 105, 313–327, doi:10.1039/ct9140500313.
[41]
Grignon, J.; Scallan, A.M. Effect of pH and neutral salts upon the swelling of cellulose gels. J. Appl. Polym. Sci. 1980, 25, 2829–2843, doi:10.1002/app.1980.070251215.
[42]
Galaev, I.Y.; Mattiasson, B. ‘Smart’ polymers and what they could do in biotechnology and medicine. Trends Biotech. 1999, 8, 335–340, doi:10.1016/S0167-7799(99)01345-1.
[43]
Hirose, M.; Kwon, O.H.; Yamato, M.; Kikuchi, A.; Okano, T. Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. Biomacromolecules 2000, 1, 377–381, doi:10.1021/bm0002961.
[44]
Tanga, Z.; Kikuchi, A.; Akiyamaa, Y.; Okano, T. Novel cell sheet carriers using Polyion complex gel modified membranes for tissue engineering technology for cell sheet manipulation and transplantation. React. Funct. Polym. 2007, 67, 1388–1397, doi:10.1016/j.reactfunctpolym.2007.07.058.
[45]
Harimoto, M.; Yamato, M.; Hirose, M.; Takahashi, C.; Isoi, Y.; Kikuchi, A.; Okano, T. Novel approach for achieving double-layered cell sheets co-culture: Overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. J. Biomed. Mater. Res. 2002, 62, 464–470, doi:10.1002/jbm.10228.
[46]
Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials 2003, 24, 2309–2316, doi:10.1016/S0142-9612(03)00110-8.
[47]
Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Maeda, N.; Watanabe, H.; Yamamoto, K.; Nagai, S.; Kikuchi, A.; Tano, Y.; et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo an a temperature-responsive cell culture surface. Transplantation 2004, 77, 379–385, doi:10.1097/01.TP.0000110320.45678.30.
[48]
Akizuki, T.; Oda, S.; Komaki, M.; Tsuchioka, H.; Kawakatsu, N.; Kikuchi, A.; Yamato, M.; Okano, T.; Ishikawa, I. Application Of periodontal ligament cell sheet for periodontal regeneration: A pilot study in beagle dogs. J. Periodontal. Res. 2005, 40, 245–251, doi:10.1111/j.1600-0765.2005.00799.x.
[49]
Ide, T.; Nishida, K.; Yamato, M.; Sumide, T.; Utsumi, M.; Nozaki, T.; Kikuchi, A.; Okano, T.; Tano, Y. Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes. Biomaterials 2006, 27, 607–614, doi:10.1016/j.biomaterials.2005.06.005.
[50]
Vermette, M.; Trottier, V.; Menard, V.; Saint-Pierre, L.; Roy, A.; Fradette, J. Production of A new tissue-engineered adipose substitute from human adipose-derived stromal cells. Biomaterials 2007, 28, 2850–2860, doi:10.1016/j.biomaterials.2007.02.030.
[51]
Obokata, H.; Yamato, M.; Yang, J.; Nishida, K.; Tsuneda, S.; Okano, T. Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture dishes. J. Biomed. Mater. Res. Part A 2008, 86, 1088–1096.
[52]
Larouche, D.; Paquet, C.; Fradette, J.; Carrier, P.; Auger, F.A.; Germain, L. Regeneration of skin and cornea by tissue engineering. Meth. Mol. Biol. 2009, 482, 233–256, doi:10.1007/978-1-59745-060-7_15.
[53]
Labbe, B.; Marceau-Fortier, G.; Fradette, J. Cell sheet technology for tissue engineering: The self-assembly approach using adipose-derived stromal cells. Meth. Mol. Biol. 2011, 702, 429–441, doi:10.1007/978-1-61737-960-4_31.
[54]
Fortier, G.M.; Gauvin, R.; Proulx, M.; Vallee, M.; Fradette, J. Dynamic culture induces a cell-type dependent response impacting on the thickness of engineered constructive tissue. J. Tissue Eng. Regen. Med. 2013, 7, 292–301, doi:10.1002/term.522.
