Comparing the Effects of Two Cryoprotectant Protocols,Dimethyl-Sulfoxide (DMSO) and Glycerol,on the Recovery Rate of Cultured Keratinocytes on Amniotic Membrane
Restricted accessResearch articleFirst published online December, 2025
Comparing the Effects of Two Cryoprotectant Protocols,Dimethyl-Sulfoxide (DMSO) and Glycerol,on the Recovery Rate of Cultured Keratinocytes on Amniotic Membrane
Background: Off-the-shelf supply of viable engineered tissue is critical for effective and fast treatment of life-threatening injuries such as deep burns. An expanded keratinocyte sheet on the human amniotic membrane (KC sheet-HAM) is a beneficial tissue-engineering product for wound healing. To access an on-hand supply for the widespread application and overcome the time-consuming process, it is necessary to develop a cryopreservation protocol that guarantees the higher recovery of viable keratinocyte sheets after freeze-thawing. This research aimed to compare the recovery rate of KC sheet-HAM after cryopreservation by dimethyl-sulfoxide (DMSO) and glycerol. Methods: Amniotic membrane was decellularized with trypsin, and keratinocytes were cultured on it to form a multilayer, flexible, easy-to-handle KC sheet-HAM. The effects of 2 different cryoprotectants were investigated by histological analysis, live-dead staining, and proliferative capacity assessments before and after cryopreservation. Results: KCs well adhered and proliferated on the decellularized amniotic membrane and successfully represented 3 to 4 stratified layers of epithelialization after 2 to 3 weeks culture period; making it easy to cut, transfer, and cryopreserve. However, viability and proliferation assay indicated that both DMSO and glycerol cryosolutions have detrimental effects on KCs, and KCs-sheet HAM could not recover to the control level after 8 days of culture post-cryo. The KC sheet lost its stratified multilayer nature on AM, and sheet layers were reduced in both cryo-groups compared to the control. Conclusion: Expanding keratinocytes on the decellularized amniotic membrane as a multilayer sheet made a viable easy-to-handle sheet, nonetheless cryopreservation reduced viability and affected histological structure after thawing. Although some viable cells were detectable, our research highlighted the need for a better cryoprotectant protocol other than DMSO and glycerol, specific for the successful banking of viable tissue constructs.
RheinwaldJGGreenH. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell. 1975;6(3):317–330.
2.
O’ConnorNMullikenJBanks-SchlegelSKehindeOGreenH. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet (London, England). 1981;1(8211):75–78.
3.
SoejimaKShimodaKKashimuraT, et al. Wound dressing material containing lyophilized allogeneic cultured cells. Cryobiology. 2013;66(3):210–214.
4.
CroninHGoldsteinG. Biologic skin substitutes and their applications in dermatology. Dermatol Surg. 2013;39(1 Pt 1):30–34.
5.
LootensLBrusselaersNBeeleHMonstreyS. Keratinocytes in the treatment of severe burn injury: an update. Int Wound J. 2013;10(1):6–12.
6.
De LucaMAlbaneseECanceddaR, et al. Treatment of leg ulcers with cryopreserved allogeneic cultured epithelium. A multicenter study. Arch Dermatol. 1992;128(5):633–638.
7.
SchiozerWAGemperliRMühlbauerWMunhozAMFerreiraMC. An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: assessment of the conservation of transplantable human skin allografts. Acta Cir Bras. 2013;28(12):824–832.
8.
YanagaHUdohYYamamotoM, et al. Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: early wound closure and suppression of scarring. Regen Ther. 2017;6:74–82.
9.
PhillipsTJProvanAColbertDEasleyKW. A randomized single-blind controlled study of cultured epidermal allografts in the treatment of split-thickness skin graft donor sites. Arch Dermatol. 1993;129(7):879–882.
10.
Bolívar-FloresYJKuri-HarcuchW. Frozen allogeneic human epidermal cultured sheets for the cure of complicated leg ulcers. Dermatol Surg. 1999;25(8):610–617.
11.
YanagaHUdohYYamauchiT, et al. Cryopreserved cultured epidermal allografts achieved early closure of wounds and reduced scar formation in deep partial-thickness burn wounds (DDB) and split-thickness skin donor sites of pediatric patients. Burns. 2001;27(7):689–698.
12.
LaroucheDCantin-WarrenLDesgagnéM, et al. Improved methods to produce tissue-engineered skin substitutes suitable for the permanent closure of full-thickness skin injuries. Biores Open Access. 2016;5(1):320–329.
13.
KatoHMarceloCLWashingtonJBBinghamELFeinbergSE. Fabrication of large size ex vivo- produced oral mucosal equivalents for clinical application. Tissue Eng Part C Methods. 2015;21(9):872–880.
14.
McheikJNBarraultCPedrettiN, et al. Foreskin-isolated keratinocytes provide successful extemporaneous autologous paediatric skin grafts. J Tissue Eng Regen Med. 2013;10(3):252–260.
15.
AtiyehBSCostagliolaM. Cultured epithelial autograft (CEA) in burn treatment: three decades later. Burns. 2007;33(4):405–413.
16.
RezaieFMomeni-MoghaddamMNaderi-MeshkinH. Regeneration and repair of skin wounds: various strategies for treatment. Int J Low Extrem Wounds. 2019;18(3):247–261.
17.
GottipamulaSSaraswatSKSridharKN. Comparative study of isolation, expansion and characterization of epithelial cells. Cytotherapy. 2017;19(2):263–271.
18.
MaarofMLawJXChowdhurySRKhairojiKABinSaim AIdrusRBH. Secretion of wound healing mediators by single and bi-layer skin substitutes. Cytotechnology. 2016;68(5):1873–1884.
19.
PetrofGAbdul-WahabAMcGrathJA. Cell therapy in dermatology. Cold Spring Harb Perspect Med. 2014;4(6):1–29.
20.
IlicDVicovacLNikolicMLazic IlicE. Human amniotic membrane grafts in therapy of chronic non-healing wounds. Br Med Bull. 2016;117(1):59–67.
21.
MamedeACCarvalhoMJAbrantesAMLaranjoMMaiaCJBotelhoMF. Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res. 2012;349(2):447–458.
22.
GrueterichMEspanaEMTouhamiATiSETsengSCG. Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency. Ophthalmology. 2002;109(8):1547–1552.
23.
GrueterichMEspanaEMTsengSCG. Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche. Surv Ophthalmol. 2003;48(6):631–646.
24.
TsaiRJFLiLChenJLadasJGStarkW. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2001;2(1):18–19.
TauzinHRolinGViennetCSaasPHumbertPMuretP. A skin substitute based on human amniotic membrane. Cell Tissue Bank. 2014;15(2):257–265.
27.
ShivakumarSBBhartiDSubbaraoRB, et al. DMSO- and Serum-free cryopreservation of Wharton’s jelly tissue isolated from human umbilical cord. J Cell Biochem. 2016;117(10):2397–2412.
28.
JacksonCEidetJRReppeS, et al. Effect of storage temperature on the phenotype of cultured epidermal cells stored in xenobiotic-free medium. Curr Eye Res. 2016;41(6):757–768.
29.
ChenFZhangWWuW, et al. Cryopreservation of tissue-engineered epithelial sheets in trehalose. Biomaterials. 2011;32(33):8426–8435.
30.
NiknejadHDeihimTPeiroviHAbolghasemiH. Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold. Cryobiology. 2013;67(1):56–63.
31.
PromprasitDBumroongkitKTocharusCMevateeUTananuvatN. Cultivation and phenotypic characterization of rabbit epithelial cells expanded ex vivo from fresh and cryopreserved limbal and oral mucosal explants. Curr Eye Res. 2015;40(3):274–281.
32.
MerymanHT. Cryopreservation of living cells: principles and practice. Transfusion. 2007;47(5):935–945.
33.
UdohYYanagaHTaiYKiyokawaKInoueY. Long-term viability of cryopreserved cultured epithelial grafts. Burns. 2000;26(6):535–542.
34.
NaaldijkYFriedrich-StöckigtASetheSStolzingA. Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation. J Tissue Eng Regen Med. 2016;10(10):E354–E364.
35.
ToosiSNaderi-MeshkinHKalaliniaF, et al. PGA-incorporated collagen: toward a biodegradable composite scaffold for bone-tissue engineering. J Biomed Mater Res - Part A. 2016;104(8):2020–2028.
36.
CoolenNAVerkerkMReijnenL, et al. Culture of keratinocytes for transplantation without the need of feeder layer cells. Cell Transplant. 2007;16(6):649–661.
37.
NakamuraTEndoK-ICooperLJ, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Investig Opthalmology Vis Sci. 2003;44(1):106.
38.
LlamesSGarcía-PérezEMeanaÁ, et al. Feeder layer cell actions and applications. Tissue Eng Part B Rev. 2015;21(4):345–353.
39.
YehH-JYaoC-LChenH-IChengH-CHwangS-M. Cryopreservation of human limbal stem cells ex vivo expanded on amniotic membrane. Cornea. 2008;27(3):327–333.
40.
TjinMSChuaAWCMoreno-MoralA, et al. Biologically relevant laminin as chemically defined and fully human platform for human epidermal keratinocyte culture. Nat Commun. 2018;9(1):4432.
41.
HäkkinenLKoivistoLLarjavaH. An improved method for culture of epidermal keratinocytes from newborn mouse skin. Methods Cell Sci. 2001;23(4):189–196.
42.
ZhangLJ. Isolation, culture, and characterization of primary mouse epidermal keratinocytes. In: Methods in Molecular Biology. 2019;1940:205–215.
43.
HuangGJiSLuoP, et al. Accelerated expansion of epidermal keratinocyte and improved dermal reconstruction achieved by engineered amniotic membrane. Cell Transplant. 2013;22(10):1831–1844.
44.
Mahmoudi-RadMAbolhasaniEMoravvejHMahmoudi-RadNMirdamadiY. Acellular amniotic membrane: an appropriate scaffold for fibroblast proliferation. Clin Exp Dermatol. 2013;38(6):646–651.
45.
YangLShirakataYTokumaruS, et al. Living skin equivalents constructed using human amnions as a matrix. J Dermatol Sci. 2009;56(3):188–195.
46.
HassanzadehHMatinMMNaderi-MeshkinH, et al. Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications. Cell Tissue Bank. 2018;19(4):531–547.
47.
MazurP. Freezing of living cells: Mechanisms and implications. Am J Physiol. 1984;247(3 Pt 1):C125 –C142.
