Effects of storage culture media, temperature and duration on human adipose‑derived stem cell viability for clinical use

Wu,Yin‑Di; Li,Meng; Liao,Xuan; Li,Sheng‑Hong; Yan,Jian‑Xin; Fan,Lei; She,Wen‑Li; Song,Jian‑Xin; Liu,Hong‑Wei

doi:10.3892/mmr.2019.9842

Effects of storage culture media, temperature and duration on human adipose‑derived stem cell viability for clinical use

Authors:
- Yin‑Di Wu
- Meng Li
- Xuan Liao
- Sheng‑Hong Li
- Jian‑Xin Yan
- Lei Fan
- Wen‑Li She
- Jian‑Xin Song
- Hong‑Wei Liu
View Affiliations

Affiliations: Department of Plastic Surgery, The First Affiliated Hospital of Jinan University, Key Laboratory of Regenerative Medicine, Ministry of Education, Guangzhou, Guangdong 510630, P.R. China, Department of Orthopedics, Third Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510630, P.R. China, Department of Plastic Surgery, Changhai Hospital, The Second Military Medical University, Shanghai 200433, P.R. China
Published online on: January 10, 2019 https://doi.org/10.3892/mmr.2019.9842
Pages: 2189-2201
Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Adipose‑derived stem cells (ADSCs) are mesenchymal stem cells that are often used in regenerative medicine. Maintaining ADSC viability is important, as this optimizes the curative effects of cell therapy. However, the optimal conditions for cell viability preservation remain unknown. The present study aimed to acquire a better protocol for ADSC storage by comparing the effects of various solutions and temperatures for ADSC preservation, in order to suggest the most effective methods of short‑term ADSC preservation for clinical use. ADSCs from passage 2 were suspended in solutions comprising 0.9% NaCl, 10% human serum (HS) or 10% platelet‑rich plasma (PRP). Suspended cells were maintained at 4˚C or room temperature (~26˚C) for 2, 4 and 6 h. The differentiation capacity, apoptosis and proliferation of ADSCs were determined by oil red O/alizarin red S staining, flow cytometry, and a cell counting kit‑8 cell proliferation assay, respectively. In addition, reverse transcription‑quantitative polymerase chain reaction and western blot analysis was performed. The results revealed that proliferation of ADSCs decreased with time. The optimal time for ADSC use was ~2 h, and 4 h was determined to be the latest time that ADSCs should be used. The 10% HS group had the highest survival rate, followed by the 10% PRP group; these two groups had higher survival rates than the 0.9% NaCl group (P<0.05). HS and PRP at 4˚C enhanced the ADSC proliferation rate (P<0.05), although the difference between these two groups was insignificant (P>0.05). In conclusion, the optimal time to use ADSCs was <2 h, and should not exceed 4 h. It was recommended that, for the transportation and short‑term storage of ADSCs during clinical use, they should be stored with 10% HS at 4˚C to maintain ADSC viability. In addition, this was a cost‑effective and safe method.

View Figures

View References

1	Gimble JM, Katz AJ and Bunnell BA: Adipose-derived stem cell for regenerative medicine. Circ Res. 100:1249–1260. 2007. View Article : Google Scholar : PubMed/NCBI
2	Bora P and Majumdar AS: Adipose tissue-derived stromal vascular fraction in regenerative medicine: A brief review on biology and translation. Stem Cell Res Ther. 8:1452017. View Article : Google Scholar : PubMed/NCBI
3	Gimble JM: Adipose tissue-derived therapeutics. Expert Opin Biol Ther. 3:705–713. 2003. View Article : Google Scholar : PubMed/NCBI
4	Baer PC and Geiger H: Adipose-derived mesenchymal stromal/stem cell: Tissue localization, characterization, and heterogeneity. Stem Cells Int 2012. 8126932012.
5	Qiu X, Fandel TM, Ferretti L, Albersen M, Orabi H, Zhang H, Lin G, Lin CS, Schroeder T and Lue TF: Both immediate and delayed intracavernous injection of autologous adipose-derived stromal vascular fraction enhances recovery of erectile function in a rat model of cavernous nerve injury. Eur Urol. 62:720–727. 2012. View Article : Google Scholar : PubMed/NCBI
6	Lin G, Garcia M, Ning H, Banie L, Guo YL, Lue TF and Lin CS: Defining stem and progenitor cell within adipose tissue. Stem Cells Dev. 17:1053–1063. 2008. View Article : Google Scholar : PubMed/NCBI
7	Tobita M, Orbay H and Mizuno H: Adipose-derived stem cell: Current findings and future perspectives. Discov Med. 11:160–170. 2011.PubMed/NCBI
8	Gonda K, Shigeura T, Sato T, Matsumoto D, Suga H, Inoue K, Aoi N, Kato H, Sato K, Murase S, et al: Preserved proliferative capacity and multipotency of human adipose-derived stem cell after long-term cryopreservation. Plast Reconstr Surg. 121:401–410. 2008. View Article : Google Scholar : PubMed/NCBI
9	Gonzalez-Fernandez ML, Perez-Castrillo S, Ordas-Fernandez P, Lopez-Gonzalez ME, Colaco B and Villar-Suarez V: Study on viability and chondrogenic differentiation of cryopreserved adipose tissue-derived mesenchymal stromal cell for future use in regenerative medicine. Cryobiology. 71:256–263. 2015. View Article : Google Scholar : PubMed/NCBI
10	Matsumoto D, Shigeura T, Sato K, Inoue K, Suga H, Kato H, Aoi N, Murase S, Gonda K and Yoshimura K: Influences of preservation at various temperatures on liposuction aspirates. Plast Reconstr Surg. 120:1510–1517. 2007. View Article : Google Scholar : PubMed/NCBI
11	Saragusty J and Arav A: Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 141:1–19. 2011. View Article : Google Scholar : PubMed/NCBI
12	Saragusty J, Gacitua H, Rozenboim I and Arav A: Protective effects of iodixanol during bovine sperm cryopreservation. Theriogenology. 71:1425–1432. 2009. View Article : Google Scholar : PubMed/NCBI
13	Bunnell BA, Flaat M, Gagliardi C, Patel B and Ripoll C: Adipose-derived stem cell: Isolation, expansion and differentiation. Methods. 45:115–120. 2008. View Article : Google Scholar : PubMed/NCBI
14	Zhang F, Ren H, Shao X, Zhuang C, Chen Y and Qi N: Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application. PeerJ. 5:e33012017. View Article : Google Scholar : PubMed/NCBI
15	Higman MA, Port JD, Beauchamp NJ Jr and Chen AR: Reversible leukoencephalopathy associated with re-infusion of DMSO preserved stem cell. Bone Marrow Transplant. 26:797–800. 2000. View Article : Google Scholar : PubMed/NCBI
16	Liu Y, Zhou Y, Feng H, Ma GE and Ni Y: Injectable tissue-engineered bone composed of human adipose-derived stromal cell and platelet-rich plasma. Biomaterials. 29:3338–3345. 2008. View Article : Google Scholar : PubMed/NCBI
17	Shafaei H, Esmaeili A, Mardani M, Razavi S, Hashemibeni B, Nasr-Esfahani MH, Shiran MB and Esfandiari E: Effects of human placental serum on proliferation and morphology of human adipose tissue-derived stem cell. Bone Marrow Transplant. 46:1464–1471. 2011. View Article : Google Scholar : PubMed/NCBI
18	Jalowiec JM, D'Este M, Bara JJ, Denom J, Menzel U, Alini M, Verrier S and Herrmann M: An in vitro investigation of platelet-rich plasma-gel as a cell and growth factor delivery vehicle for tissue engineering. Tissue Eng Part C Methods. 22:49–58. 2016. View Article : Google Scholar : PubMed/NCBI
19	Weibrich G, Kleis WK, Hafner G and Hitzler WE: Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg. 30:97–102. 2002. View Article : Google Scholar : PubMed/NCBI
20	Freymann U, Degrassi L, Kruger JP, Metzlaff S, Endres M and Petersen W: Effect of serum and platelet-rich plasma on human early or advanced degenerative meniscus cells. Connect Tissue Res. 58:509–519. 2017. View Article : Google Scholar : PubMed/NCBI
21	Bura A, Planat-Benard V, Bourin P, Silvestre JS, Gross F, Grolleau JL, Saint-Lebese B, Peyrafitte JA, Fleury S, Gadelorge M, et al: Phase I trial: The use of autologous cultured adipose-derived stroma/stem cell to treat patients with non-revascularizable critical limb ischemia. Cytotherapy. 16:245–257. 2014. View Article : Google Scholar : PubMed/NCBI
22	Guo X, Li S, Ji Q, Lian R and Chen J: Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture. Hum Cell. 28:175–189. 2015. View Article : Google Scholar : PubMed/NCBI
23	Gu H, Guo F, Zhou X, Gong L, Zhang Y, Zhai W, Chen L, Cen L, Yin S, Chang J and Cui L: The stimulation of osteogenic differentiation of human adipose-derived stem cell by ionic products from akermanite dissolution via activation of the ERK pathway. Biomaterials. 32:7023–7033. 2011. View Article : Google Scholar : PubMed/NCBI
24	Wang J, Ye Y, Tian H, Yang S, Jin X, Tong W and Zhang Y: In vitro osteogenesis of human adipose-derived stem cell by coculture with human umbilical vein endothelial cell. Biochem Biophys Res Commun. 412:143–149. 2011. View Article : Google Scholar : PubMed/NCBI
25	Visweswaran M, Schiefer L, Arfuso F, Dilley RJ, Newsholme P and Dharmarajan A: Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cell. PLoS One. 10:e1180052015. View Article : Google Scholar
26	Li HX, Luo X, Liu RX, Yang YJ and Yang GS: Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cell. Mol Cell Endocrinol. 291:116–124. 2008. View Article : Google Scholar : PubMed/NCBI
27	Samuel S, Ahmad RE, Ramasamy TS, Karunanithi P, Naveen SV, Murali MR, Abbas AA and Kamarul T: Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cell. PeerJ. 4:e23472016. View Article : Google Scholar : PubMed/NCBI
28	Bunnell BA, Estes BT, Guilak F and Gimble JM: Differentiation of adipose stem cell. Methods Mol Biol. 456:155–171. 2008. View Article : Google Scholar : PubMed/NCBI
29	Wang X, Spandidos A, Wang H and Seed B: PrimerBank: A PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res. 40:D1144–D1149. 2012. View Article : Google Scholar : PubMed/NCBI
30	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
31	Frese L, Dijkman PE and Hoerstrup SP: Adipose tissue-derived stem cells in regenerative medicine. Transfus Med Hemother. 43:268–274. 2016. View Article : Google Scholar : PubMed/NCBI
32	Martínez-González I, Cruz MJ, Moreno R, Morell F, Muñoz X and Aran JM: Human mesenchymal stem cell resolve airway inflammation, hyperreactivity, and histopathology in a mouse model of occupational asthma. Stem Cells Dev. 23:2352–2363. 2014. View Article : Google Scholar : PubMed/NCBI
33	Won CH, Yoo HG, Kwon OS, Sung MY, Kang YJ, Chung JH, Park BS, Sung JH, Kim WS and Kim KH: Hair growth promoting effects of adipose tissue-derived stem cell. J Dermatol Sci. 57:134–137. 2010. View Article : Google Scholar : PubMed/NCBI
34	Hernández-Bule ML, Martínez-Botas J, Trillo MÁ, Paíno CL and Úbeda A: Antiadipogenic effects of subthermal electric stimulation at 448 kHz on differentiating human mesenchymal stem cells. Mol Med Rep. 13:3895–3903. 2016. View Article : Google Scholar : PubMed/NCBI
35	Rumiński S, Ostrowska B, Jaroszewicz J, Skirecki T, Włodarski K, Święszkowski W and Lewandowska-Szumieł M: Three-dimensional printed polycaprolactone-based scaffolds provide an advantageous environment for osteogenic differentiation of human adipose-derived stem cells. J Tissue Eng Regen Med. 12:e473–e485. 2018. View Article : Google Scholar : PubMed/NCBI
36	Benazzo F, Botta L, Scaffino MF, Caliogna L, Marullo M, Fusi S and Gastaldi G: Trabecular titanium can induce in vitro osteogenic differentiation of human adipose derived stem cells without osteogenic factors. J Biomed Mater Res A. 102:2061–2071. 2014. View Article : Google Scholar : PubMed/NCBI
37	Rada T, Reis RL and Gomes ME: Distinct stem cells subpopulations isolated from human adipose tissue exhibit different chondrogenic and osteogenic differentiation potential. Stem Cell Rev. 7:64–76. 2011. View Article : Google Scholar : PubMed/NCBI
38	Smyth DC, Takenaka S, Yeung C and Richards CD: Oncostatin M regulates osteogenic differentiation of murine adipose-derived mesenchymal progenitor cells through a PKCdelta-dependent mechanism. Cell Tissue Res. 360:309–319. 2015. View Article : Google Scholar : PubMed/NCBI
39	Regassa A, Suh M, Datar J, Chen C and Kim WK: Fatty acids have different adipogenic differentiation potentials in stromal vascular cells isolated from abdominal fat in laying hens. Lipids. 52:513–522. 2017. View Article : Google Scholar : PubMed/NCBI
40	Nurden AT, Nurden P, Sanchez M, Andia I and Anitua E: Platelets and wound healing. Front Biosci. 13:3532–3548. 2008.PubMed/NCBI
41	Whitman DH, Berry RL and Green DM: Platelet gel: An autologous alternative to fibrin glue with applications in oral and maxillofacial surgery. J Oral Maxillofac Surg. 55:1294–1299. 1997. View Article : Google Scholar : PubMed/NCBI
42	Alsousou J, Thompson M, Hulley P, Noble A and Willett K: The biology of platelet-rich plasma and its application in trauma and orthopaedic surgery: A review of the literature. J Bone Joint Surg Br. 91:987–996. 2009. View Article : Google Scholar : PubMed/NCBI
43	Nikolidakis D and Jansen JA: The biology of platelet-rich plasma and its application in oral surgery: Literature review. Tissue Eng Part B Rev. 14:249–258. 2008. View Article : Google Scholar : PubMed/NCBI
44	Anitua E: Plasma rich in growth factors: Preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants. 14:529–535. 1999.PubMed/NCBI
45	Drago L, Bortolin M, Vassena C, Romanò CL, Taschieri S and Del Fabbro M: Plasma components and platelet activation are essential for the antimicrobial properties of autologous platelet-rich plasma: An in vitro study. PLoS One. 9:e1078132014. View Article : Google Scholar : PubMed/NCBI
46	Margolis DJ, Kantor J, Santanna J, Strom BL and Berlin JA: Effectiveness of platelet releasate for the treatment of diabetic neuropathic foot ulcers. Diabetes Care. 24:483–488. 2001. View Article : Google Scholar : PubMed/NCBI
47	Josh F, Kobe K, Tobita M, Tanaka R, Suzuki K, Ono K, Hyakusoku H and Mizuno H: Accelerated and safe proliferation of human adipose-derived stem cell in medium supplemented with human serum. J Nippon Med Sch. 79:444–452. 2012. View Article : Google Scholar : PubMed/NCBI
48	Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Smith RD, Springer DL and Pounds JG: Toward a human blood serum proteome: Analysis by multidimensional separation coupled with mass spectrometry. Mol Cell Proteomics. 1:947–955. 2002. View Article : Google Scholar : PubMed/NCBI
49	Kobayashi T, Watanabe H, Yanagawa T, Tsutsumi S, Kayakabe M, Shinozaki T, Higuchi H and Takagishi K: Motility and growth of human bone-marrow mesenchymal stem cell during ex vivo expansion in autologous serum. J Bone Joint Surg Br. 87:1426–1433. 2005. View Article : Google Scholar : PubMed/NCBI