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Brazilian journal of veterinary medicine2023; 45; e002523; doi: 10.29374/2527-2179.bjvm002523

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

Abstract: The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS. As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.
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Publication Date: 2023-12-27 PubMed ID: 38162818PubMed Central: PMC10756151DOI: 10.29374/2527-2179.bjvm002523Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigated the use of different freezing solutions, including the addition of a synthetic polymer called SuperCool X-1000, to preserve equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) during cryopreservation, with the goal of reducing the concentration of the commonly used but potentially harmful cryoprotectant DMSO.
  • The research evaluated cell viability, colony formation, and differentiation capacity after freezing with various freezing solutions to assess if synthetic polymers could partially replace or reduce DMSO usage without compromising cell quality.

Background and Purpose

  • Stem cell therapies often rely on cryopreservation to store cells for long periods.
  • Dimethylsulfoxide (DMSO) is the most commonly used permeating cryoprotectant but can cause severe adverse effects when used in high concentrations.
  • The study aimed to explore whether adding non-permeating cryoprotectants, specifically Trehalose and the synthetic polymer SuperCool X-1000, could maintain stem cell quality while reducing DMSO concentration.
  • The focus was on equine adipose tissue-derived mesenchymal stem cells (eAT-MSCs), important for regenerative therapies in veterinary medicine.

Experimental Design

  • eAT-MSCs were categorized as fresh (control) or subjected to slow freezing with seven different freezing solutions (FS):
    • DMSO alone
    • Trehalose (T) alone
    • SuperCool X-1000 (X) alone
    • DMSO + Trehalose (DMSO+T)
    • DMSO + SuperCool X-1000 (DMSO+X)
    • Trehalose + SuperCool X-1000 (T+X)
    • DMSO + Trehalose + SuperCool X-1000 (DMSO+T+X)
  • Outcomes measured before and after freezing included:
    • Cell viability
    • Colony forming units (CFUs), indicating the capacity for cell proliferation
    • Cellular differentiation potential into osteogenic (bone), chondrogenic (cartilage), and adipogenic (fat) lineages

Key Findings

  • Viability:
    • Cell viability decreased after freezing in all treatments compared to fresh cells.
    • DMSO alone showed the highest post-thaw viability (80.3 ± 0.6%), significantly better than other freezing solutions.
  • Colony Forming Units (CFUs):
    • No significant differences were found between fresh and frozen cells across treatments, indicating preserved proliferation capacity.
  • Differentiation capacity:
    • Frozen cells maintained the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages.
    • Osteogenic differentiation was significantly reduced in frozen cells compared to fresh but did not differ statistically across different freezing solutions.
    • Chondrogenic differentiation was significantly lower in cells frozen with DMSO + SuperCool X-1000 compared to control but was not statistically different from other freezing solutions.
    • Adipogenic differentiation showed no significant differences among treatments.

Interpretation and Implications

  • The study confirmed that DMSO remains the most effective single cryoprotectant for preserving viability and function of equine adipose-derived MSCs after slow freezing.
  • The synthetic polymer SuperCool X-1000 showed promise as an additive that could potentially reduce the concentration of DMSO needed in freezing solutions without significantly impairing key cell characteristics.
  • This is important because lowering DMSO concentration could reduce its toxic side effects in clinical or veterinary regenerative therapies.
  • Further research is required to optimize the concentrations and combinations of these cryoprotectants to improve frozen stem cell quality while minimizing adverse effects.

Conclusion

  • While DMSO-based freezing solutions are currently superior for cryopreserving equine adipose tissue-derived MSCs, the inclusion of SuperCool X-1000 as a synthetic polymer additive shows potential to decrease the reliance on DMSO.
  • This approach could lead to safer and more effective storage methods for stem cells intended for regenerative therapies.

Cite This Article

APA
Nascimento C, Saraiva MVA, Pereira VM, de Brito DCC, de Aguiar FLN, Alves BG, Roballo KCS, de Figueiredo JR, Ambrósio CE, Rodrigues APR. (2023). Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration. Braz J Vet Med, 45, e002523. https://doi.org/10.29374/2527-2179.bjvm002523

Publication

Brazilian journal of veterinary medicine
ISSN: 2527-2179
NlmUniqueID: 9918435088106676
Country: Brazil
Language: English
Volume: 45
Pages: e002523
PII: e002523

Researcher Affiliations

Nascimento, Cátia
  • Veterinarian, MSc. Laboratório de Manipulação de Oócitos e Folículos Pré-Antrais Ovarianos (LAMOFOPA), Faculdade de Medicina Veterinária, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.
Saraiva, Márcia Viviane Alves
  • Veterinarian, DSc. Universidade Federal Rural do Semiárido (UFERSA), Mossoró, RN, Brazil.
Pereira, Vitoria Mattos
  • Veterinarian, MSc. Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, SP, Brazil.
de Brito, Danielle Cristina Calado
  • Biologist, DSc. LAMOFOPA, Faculdade de Medicina Veterinária, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.
de Aguiar, Francisco Léo Nascimento
  • Veterinarian, DSc. Instituto Federal de Educação, Ciência e Tecnologia da Paraíba, Sousa, PB, Brazil.
Alves, Benner Geraldo
  • Veterinarian, DSc. Laboratório de Biologia da Reprodução, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
Roballo, Kelly Cristine Santos
  • Veterinarian, DSc. Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, SP, Brazil.
de Figueiredo, José Ricardo
  • Veterinarian, DSc. LAMOFOPA, Faculdade de Medicina Veterinária, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.
Ambrósio, Carlos Eduardo
  • Veterinarian, DSc. Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, SP, Brazil.
Rodrigues, Ana Paula Ribeiro
  • Veterinarian, DSc. LAMOFOPA, Faculdade de Medicina Veterinária, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.

Conflict of Interest Statement

Conflict of interests: No conflict of interest.

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