Cryopreservation of adipose tissue-derived mesenchymal stem cells: An alternative to serial fat grafting in breast reconstruction

▪ ABSTRACT

Introduction:

Fat grafting is an autologous graft of cells from subcutaneous tissue, which can be used as a complementary technique in breast reconstruction. Given this, the cryopreservation of adipose tissue-derived mesenchymal stem cells (ADMSCs) could be a way to collect them in one surgical procedure and after performing fractional fat grafting. Dimethyl sulfoxide (DMSO) is a cryopreservative used in cell research, but it is potentially toxic, which would make it impossible to use cryopreserved ADMSCs in clinical practice. New cellular cryopreservatives, without toxicity, have been described in the experimental scientific literature, such as the substances L-proline and trehalose. Therefore, this work aimed to evaluate the viability of ADMSCs cryopreserved with the combination of L-proline and trehalose over up to 90 days.

Method:

Experimental study in which lipoaspirate samples were obtained from 9 patients. The cellular fraction was processed and frozen with L-proline (1.5M) + trehalose (0.2M) or with DMSO + fetal bovine serum (FBS) as control. After 30 and 90 days, the samples were thawed, and cell viability was assessed using the MTT technique.

Results:

The analysis of ADMSCs, after 1 and 3 months of freezing, indicated that samples treated with L-proline + trehalose showed similar viability to those preserved with DMSO and SFB (p=0.444).

Conclusion:

The association of L-proline and trehalose kept ADMSC viable for 30 and 90 days of freezing, and could be an alternative as a cellular cryopreservative without toxicity and enabling the use of serial fat grafting.

Keywords:

Trehalose - Lipectomy - Breast neoplasms - Mesenchymal stem cells - Adipocytes - Proline

Institution: Universidade Comunitária da Região de Chapecó (Unochapecó), Chapecó, SC, Brazil.

Publication History

Received: 15 May 2023

Accepted: 20 August 2023

Article published online:
20 May 2025

• REFERENCES

• 1 Fanakidou I, Zyga S, Alikari V, Tsironi M, Stathoulis J, Theofilou P. Mental health, loneliness, and illness perception outcomes in quality of life among young breast cancer patients after mastectomy: the role of breast reconstruction. Qual Life Res 2018; 27 (02) 539-543
• 2 Brasil. Presidência da República. Lei Nº 9.797, de 6 de maio de 1999. Dispõe sobre a obrigatoriedade da cirurgia plástica reparadora da mama pela rede de unidades integrantes do Sistema Único de Saúde - SUS nos casos de mutilação decorrentes de tratamento de câncer. Brasília: Presidência da República; 1999. . [Internet]. Disponível em: https://legislacao.presidencia.gov.br/atos/?tipo=LEI&numero=9797&ano=1999&ato=b04cXRE9keNpWT174
• 3 Brasil. Presidência da República. Lei Nº 12.802, de 24 de abril de 2013. Altera a Lei nº 9.797, de 6 de maio de 1999, que “dispõe sobre a obrigatoriedade da cirurgia plástica reparadora da mama pela rede de unidades integrantes do sistema único de saúde - SUS nos casos de mutilação decorrentes de tratamento de câncer”, para dispor sobre o momento da reconstrução mamária. [Internet]. Brasília: Presidência da República; 2013. . Disponível em: https://legislacao.presidencia.gov.br/atos/?tipo=LEI&numero=12802&ano=2013&ato=97cATRU50MVpWTabf
• 4 Brasil. Ministério da Saúde. Portaria GM/MS nº 127, de 13 de fevereiro de 2023. Institui estratégia excepcional de ampliação do acesso à reconstrução mamária em caso de mulheres com diagnóstico de câncer de mama, no âmbito do Sistema Único de Saúde -SUS. [Internet]. Brasília: Ministério da Saúde; 2023. . Disponível em: https://www.gov.br/saude/pt-br/composicao/saes/legislacao/portaria-gm-ms-no-127-de-13-de-fevereiro-de-2023/view
• 5 Loibl S, Poortmans P, Morrow M, Denkert C, Curigliano G. Breast cancer. Lancet 2021; 397 (10286): 1750-1769
• 6 Petrucelli N, Daly MB, Pal T. BRCA1- and BRCA2-Associated Hereditary Breast and Ovarian Cancer: Synonym: BRCA1- and BRCA2-Associated HBOC [Internet]. 2022 [acesso 2022 nov 11]. Disponível em: https://www.ncbi.nlm.nih.gov/books/NBK1247/
• 7 Tan SS, Loh W. The utility of adipose-derived stem cells and stromal vascular fraction for oncologic soft tissue reconstruction: Is it safe? A matter for debate. Surgeon 2017; 15 (04) 186-189
• 8 Stumpf CC, Zucatto ÂE, Cavalheiro JAC, de Melo MP, Cericato R, Damin APS. et al. Oncologic safety of immediate autologous fat grafting for reconstruction in breast-conserving surgery. Breast Cancer Res Treat 2020; 180 (02) 301-309
• 9 Krastev TK, Schop SJ, Hommes J, Piatkowski AA, Heuts EM, van der Hulst RRWJ. Meta-analysis of the oncological safety of autologous fat transfer after breast cancer. Br J Surg 2018; 105 (09) 1082-1097
• 10 He X, Zhang J, Luo L, Shi J, Hu D. New Progress of Adipose-derived Stem Cells in the Therapy of Hypertrophic Scars. Curr Stem Cell Res Ther 2020; 15 (01) 77-85
• 11 Zuk P. Adipose-Derived Stem Cells in Tissue Regeneration: A Review. Int Sch Res Notices 2013; 2013: 1-35
• 12 Moreno M, Schmidt JC, Gazzoni CD, Dal-Magro L, Bonadiman BDSR, Kosvoski GC. et al. Viability of mesenchymal stem cells of adipose tissue from human liposuction. Rev Bras Cir Plást 2021; 36 (01) 9-14
• 13 da Silva A, do Prado J, Pignataro J, Moreno M. Lipoenxertia. Bagnoli F, Postiglione F, Palermo F, Pedrini JL, Freitas Júnior R, Marques V. Mastologia: do diagnóstico ao tratamento. 2ª. Goiânia: Conexão Soluções Corporativas; 2022
• 14 Arshad Z, Karmen L, Choudhary R, Smith JA, Branford OA, Brindley DA. et al. Cell assisted lipotransfer in breast augmentation and reconstruction: A systematic review of safety, efficacy, use of patient reported outcomes and study quality. JPRAS Open 2016; 10: 5-20
• 15 Piffer A, Aubry G, Cannistra C, Popescu N, Nikpayam M, Koskas M. et al. Breast Reconstruction by Exclusive Lipofilling after Total Mastectomy for Breast Cancer: Description of the Technique and Evaluation of Quality of Life. J Pers Med 2022; 12 (02) 153
• 16 Goh BC, Thirumala S, Kilroy G, Devireddy RV, Gimble JM. Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability. J Tissue Eng Regen Med 2007; 1 (04) 322-324
• 17 Lee JE, Kim I, Kim M. Adipogenic differentiation of human adipose tissue-derived stem cells obtained from cryopreserved adipose aspirates. Dermatol Surg 2010; 36 (07) 1078-1083
• 18 Wang C, Xiao R, Cao YL, Yin HY. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells. Biochem Biophys Res Commun 2017; 491 (01) 198-203
• 19 Kim IH, Yang JD, Lee DG, Chung HY, Cho BC. Evaluation of centrifugation technique and effect of epinephrine on fat cell viability in autologous fat injection. Aesthet Surg J 2009; 29 (01) 35-39
• 20 Frazier TP, Gimble JM, Devay JW, Tucker HA, Chiu ES, Rowan BG. Body mass index affects proliferation and osteogenic differentiation of human subcutaneous adipose tissue-derived stem cells. BMC Cell Biol 2013; 14: 34
• 21 Efimenko AY, Kochegura TN, Akopyan ZA, Parfyonova YV. Autologous Stem Cell Therapy: How Aging and Chronic Diseases Affect Stem and Progenitor Cells. Biores Open Access 2015; 4 (01) 26-38
• 22 Zhang TY, Tan PC, Xie Y, Zhang XJ, Zhang PQ, Gao YM. et al. The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells. Stem Cell Res Ther 2020; 11 (01) 460
• 23 Toledo LS, Mauad R. Fat injection: a 20-year revision. Clin Plast Surg 2006; 33 (01) 47-53
• 24 Piccotti F, Rybinska I, Scoccia E, Morasso C, Ricciardi A, Signati L. et al. Lipofilling in Breast Oncological Surgery: A Safe Opportunity or Risk for Cancer Recurrence?. Int J Mol Sci 2021; 22 (07) 3737
• 25 Becker H, Vazquez OA, Rosen T. Cannula Size Effect on Stromal Vascular Fraction Content of Fat Grafts. Plast Reconstr Surg Glob Open 2021; 9 (03) e3471
• 26 Tong Y, Liu P, Wang Y, Geng C, Han X, Ma J. et al. The Effect of Liposuction Cannula Diameter on Fat Retention-Based on a Rheological Simulation. Plast Reconstr Surg Glob Open 2018; 6 (11) e2021
• 27 Biazus JV, Falcão CC, Parizotto AC, Stumpf CC, Cavalheiro JA, Schuh F. et al. Immediate Reconstruction with Autologous fat Transfer Following Breast-Conserving Surgery. Breast J 2015; 21 (03) 268-275
• 28 Condé-Green A, de Amorim NF, Pitanguy I. Influence of decantation, washing and centrifugation on adipocyte and mesenchymal stem cell content of aspirated adipose tissue: a comparative study. J Plast Reconstr Aesthet Surg 2010; 63 (08) 1375-1381
• 29 Coleman SR. Long-term survival of fat transplants: controlled demonstrations. Aesthetic Plast Surg 1995; 19 (05) 421-425
• 30 Ladeira PRS, Isaac C, Nakamura YM, Tutihashi RMC, Paggiaro AO, Ferreira MC. Cultivo de células-tronco derivadas de tecido adiposo: uma análise crítica. Rev Med (São Paulo) 2012; 91 (04) 246-252
• 31 Irioda AC. Avaliação da integridade das células-tronco mesenquimais derivadas do tecido adiposo humano após o bioprocesso de criopreservação [Dissertação de mestrado]. Curitiba: Universidade Federal do Paraná; 2010. . Disponível em: http://hdl.handle.net/1884/26521
• 32 De Rosa A, De Francesco F, Tirino V, Ferraro GA, Desiderio V, Paino F. et al. A new method for cryopreserving adipose-derived stem cells: an attractive and suitable large-scale and long-term cell banking technology. Tissue Eng Part C Methods 2009; 15 (04) 659-667
• 33 Ray SS, Pramanik K, Sarangi SK, Jain N. Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells. Biotechnol Lett 2016; 38 (08) 1397-1404
• 34 Ginani F, Soares DM, Barboza CAG. Influência de um protocolo de criopreservação no rendimento in vitro de células-tronco derivadas do tecido adiposo. Rev Bras Cir Plást 2012; 27 (03) 359-363
• 35 Verheijen M, Lienhard M, Schrooders Y, Clayton O, Nudischer R, Boerno S. et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci Rep 2019; 9 (01) 4641
• 36 Kennelly P, Rodwell V. Aminoácidos e Peptídeos. Rodwell VW, Bender D, Botham KM, Kennelly PJ, Weil PA. Bioquímica Ilustrada de Harper. 31ª. Porto Alegre: AMGH Editora; 2021
• 37 Dou M, Lu C, Sun Z, Rao W. Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation. Cryobiology 2019; 91: 23-29
• 38 Dovgan B, Miklavčič D, Knežević M, Zupan J, Barlič A. Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation. Cytotechnology 2021; 73 (03) 391-411
• 39 Dalmagro JP. Isolamento e caracterização das células-tronco mesenquimais provenientes do tecido adiposo e avaliação da sua viabilidade tecidual após criopreservação [Monografia]. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2019
• 40 Lenoch CY. Estratégias para criopreservação de células tronco mesenquimais de tecido adiposo bovino [Dissertação de mestrado]. Lages: Universidade do Estado de Santa Catarina; 2015
• 41 Gonda K, Shigeura T, Sato T, Matsumoto D, Suga H, Inoue K. et al. Preserved proliferative capacity and multipotency of human adipose-derived stem cells after long-term cryopreservation. Plast Reconstr Surg 2008; 121 (02) 401-410