Characterization of bovine mesenchymal stromal cells from visceral adipose tissue collected post-mortem

Caracterização de células tronco mesenquimais bovinas de tecido adiposo visceral coletadas post-mortem

ABSTRACT:

Adipose tissue can be a source of bovine mesenchymal stromal/stem cells (MSCs). However, most studies do not incorporate post-mortem collection or in vitro differentiation of these cells from bovine fat and other sources into three mesodermal lineages. This study characterized and cryopreserved bovine adipose tissue-derived MSCs (AD-MSCs) collected post-mortem. Cells were isolated from cattle (Bos taurus) and cultured under standard conditions for ex vivo expansion and cryopreservation. Cell growth kinetics was determined by cell doubling time after consecutive cellular passages. Clonogenic capacity was determined using the colony-forming unit fibroblast (CFU-F) assay. The trilineage differentiation assay was performed to determine its in vitro multipotency. The in vitro proliferation and clonogenic capacity of bovine AD-MSCs was maintained across successive passages (P₂-P₆). Greater clonogenic capacity was found in fresh than post-cryopreservation cells. Cells showed multipotential capacity in adipose, cartilage and bone lineages. In conclusion, no changes were observed in the growth kinetics and colony-forming capacity of the successive passages evaluated. Clonogenic capacity was higher in fresh than post-cryopreserved cells.

Key words:
cattle; mesenchymal stem cells; fat; cryopreservation

RESUMO:

O tecido adiposo pode ser uma fonte de células estromais/tronco mesenquimais bovinas (MSCs). No entanto, a maioria dos estudos não incorpora a coleta post-mortem ou a diferenciação in vitro dessas células a partir de gordura bovina e outras fontes em três linhagens mesodérmicas. O objetivo deste estudo foi caracterizar e criopreservar MSCs derivadas de tecido adiposo bovino (AD-MSCs) coletadas post-mortem. As células foram isoladas de bovinos (Bos taurus) e cultivadas sob condições padrão para expansão ex vivo e criopreservação. A cinética de crescimento celular foi determinada pelo tempo de duplicação celular após passagens celulares consecutivas. A capacidade clonogênica foi determinada usando o ensaio de fibroblastos de unidades formadoras de colônias (CFU-F). O ensaio de diferenciação de trilinhagem foi realizado para determinar sua multipotência in vitro. A proliferação in vitro e a capacidade clonogênica das AD-MSCs bovinas foram mantidas através de passagens sucessivas (P2-P6). Maior capacidade clonogênica foi encontrada em células frescas do que em células pós-criopreservadas. As células mostraram capacidade multipotencial nas linhagens adiposa, cartilaginosa e óssea. Concluindo, não foram observadas alterações na cinética de crescimento e na capacidade de formação de colônias das sucessivas passagens avaliadas. A capacidade clonogênica foi maior em células frescas do que em células pós-criopreservadas.

Palavras-chave:
gado; células tronco mesenquimais; tecido adiposo; criopreservação

INTRODUCTION

The possibility of ex vivo multipotency of bovine mesenchymal stromal/stem cells (MSCs), their immunomodulatory capacity and antimicrobial properties have increased scientific focus on these cells (HILL et al., 2019). There are relevant antecedents on the use of cell therapy in cattle diseases such as mastitis (CAHUASCANCO et al., 2019; LANGE-CONSIGLIO et al., 2019; PERALTA et al., 2020) and ovarian injuries caused by the follicular aspiration technique (MALARD et al., 2020). There is substantial interest in the development of “cultured meat” or “in vitro meat” from stem cells such as bovine MSCs (OKAMURA et al., 2018; RAMÍREZ-ESPINOSA et al., 2016), among other sources (SKRIVERGAARD et al., 2021).

Although, a number of studies report various aspects of bovine MSCs, few have focused on characterizing these cells. Original reports have concentrated on bone marrow (LU et al., 2014b), adipose tissue (LU et al., 2014a; SAMPAIO et al., 2015; ZHAO et al., 2012), the uterus (LARA et al., 2017) and fetal annexes, such as the placenta, umbilical cord, and amniotic fluid (RAOUFI et al., 2011; SILVA et al., 2016). However, not all characterization studies of bovine MSCs confirmed multipotency by in vitro trilineage differentiation (DOMINICI et al., 2006) of adipogenic, chondrogenic and osteogenic lines (SAMPAIO et al., 2015; ZHAO et al., 2012; ZHAO et al., 2018). In addition, only ZHAO et al. (2012) evaluated taking post-mortem samples to isolate bovine MSCs. Thus, the aim of this study was to characterize and cryopreserve bovine adipose tissue-derived MSCs (AD-MSCs) collected post-mortem. We hypothesized that collecting post-mortem samples is an advantageous strategy to isolate bovine AD-MSCs because they can be obtained from commercial abattoirs.

MATERIALS AND METHODS

Post-mortem harvesting of adipose tissue from cow carcass

Seventeen 2-month-old Holstein crossbred (Bos taurus) calves weighing 82.5-103 kgs were used for post-mortem fat harvesting. Eight-ten-gram samples were obtained from the perirenal visceral tissue (Vs).

Isolation and cryopreservation

The tissue samples were transported in a refrigerated sterile tube with low glucose (Gibco) Dulbecco’s Modified Eagle Medium (DMEM) and 2% penicillin/streptomycin (antibiotic) (Capricon). The fat was cut into small pieces, and enzymatically digested with type I collagenase (Gibco) for 40 minutes at 37 °C. The samples were centrifuged at 600g for 10 minutes, the supernatant was removed and resuspended in 10 mL of growth medium (GM) consisting of low glucose DMEM, 20% heat-inactivated fetal bovine serum (FBS) (Capricorn) and 2% antibiotic, and incubated at 37 ºC in a humidified atmosphere with 5% CO₂ (YANESELLI et al., 2018). The GM was changed every 3-4 days and 80% of confluent cells were harvested and cryopreserved in a medium composed of 95% FBS and 5% dimethyl sulfoxide (DMSO) and stored at -80 °C.

Cellular proliferation evaluation

a) Cell doubling times (DT): Post-cryopreservation bovine AD-MSCs were used to determine DT and subsequent subcultures at P₂-P₆. To that end, the cells were seeded in triplicate in a 6-well plate at a concentration of 1.7-5×10³/cm² in GM with 10% FBS under standard conditions described above. The GM was changed every 3 days. After 5-7 days, the cells were trypsinized, subcultivated and quantified by trypan blue viability. The formula used was previously described (VIDAL et al., 2006).

b) Colony-forming unit fibroblast (CFU-F) assay: Cells were seeded in triplicate in 6-well culture plates, at a concentration of 1×10³ cells/well, in GM with 10% FBS in fresh culture in P₁ and post-cryopreservation in P₂-P₆. After 7 days of culture, cells were fixed with methanol at -20°C for 15 min and stained with Giemsa for 30 min (GUERCIO et al., 2013). Colonies were macroscopically identified and counted using ImageJ software.

Trilineage differentiation assay

The cultured bovine AD-MSCs were seeded in duplicate at an initial concentration of 9.4×10³ cells/well in 24-well culture plates, and induced with the specific medium for each lineage for 21 days in GM with 10% FBS. The medium was changed every 3-4 days. Adipogenic differentiation: 5 µM isobutyl methylxanthine (Sigma), 60 µM indomethacin (Sigma), 1 µM dexamethasone (Sigma) and 50 µg/mL insulin (Gibco). Cells were then fixed with a 4% paraformaldehyde solution for 15 minutes. The presence of lipid droplets was detected by Red O staining. Chondrogenic differentiation: 1.7 mM ascorbic acid (Sigma), 10 ng/mL of TGF-β (Sigma) and 62.5 µg/mL of insulin (Gibco). The culture was fixed and stained with Alcian Blue to detect the presence of cartilaginous matrix. Osteogenic differentiation: 60 µM dexamethasone, 10 mM β-glycerophosphate (Sigma) and 1.7 mM ascorbic acid. Alizarin Red S staining was used to determine the presence of mineralized matrix. The controls were cultured in GM with 10% FBS.

Statistical analysis

DT and CFU-F in the different passages were evaluated by parametric ANOVA with Tukey’s post-hoc test. CFU-F between fresh and post-cryopreservation was compared by the t-test and the results expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant. Analyses were performed with GraphPad Prism 8 software.

RESULTS

Isolation and cryopreservation

The isolation of bovine AD-MSCs from 10 animals resulted in a success rate of 58.8 % (10/17 samples). After 24-48 h of seeding, adherent stromal cells with fibroblastic-like morphology and clonogenic capacity were observed (Figure 1). A total of 48 vials with a concentration of 1×10⁶ cells/vial were stored in the biobank.

Figure 1
Primary culture of bovine adipose tissue-derived mesenchymal stromal/stem cells (AD-MSCs) stained with Giemsa. Microscopic image of: A) primary culture showing fibroblastic-like adherence to the plastic cells and monolayer growth (10×); B) macroscopic colonies in a 6-well culture plate.

Proliferation capacity

The DT and CFU-F assays were used to determine the in vitro proliferation capacity of the AD-MSCs (n = 7). The DT assay showed P₂-P₆ subcultures with a range of 2.4 ± 0.9 to 4.4 ± 3 days (Figure 2A). There was an increase in time between P₄-P₅, albeit not significant. As determined by the CFU-F assay, clonogenic capacity varied from 24 ± 4 to 28 ± 1 colonies (Figure 2B). There were no post-cryopreservation differences between P₂-P₆. However, greater clonogenic capacity was reported in fresh cells (P₁) (74 ± 1 vs. 28 ± 1 of post-cryopreservation cells in P₂, see Figure 2C).

Figure 2
Proliferation capacity of bovine adipose tissue-derived mesenchymal stromal/stem cells (AD-MSCs). A) Cell doubling times (DT) and B) colony-forming unit fibroblast (CFU-F) were evaluated across successive passages. C) Colony-forming capacity comparison between fresh cells in P₁ and post-freezing cells in P₂ (cryopreserved). The results are expressed as mean ± SD; ^*= P < 0.05.

In vitro multipotency

To determine in vitro bovine AD-MSCs multipotency (n = 5), a trilineage differentiation assay was used. After induction, cells showed morphological and affinity changes to specific staining that indicated their differentiation to adipose (Figure 3A), cartilage (Figure 3C) and bone lineages (Figure 3E). The controls showed no affinity for any dye (Figures 3B, D and F).

Figure 3
Microscopic images for multilineages of bovine adipose tissue-derived mesenchymal stromal/stem cells (AD-MSCs). A) Adipogenic differentiation (40×) showing cell lipid droplet accumulation stained in red. C) Chondrogenic differentiation (4×) showing cartilaginous matrix stained in blue. D) Osteogenic differentiation (4×) showing mineralized matrix stained in red. B, D and F are negative controls.

DISCUSSION

In this study, we reported three important findings. First, we demonstrated for the first time, the feasibility of post-mortem isolation from cattle of Vs AD-MSCs with fibroblastic morphology and in vitro multipotency. A previous study described post-mortem isolation of these bovine cells but not from Vs fat (ZHAO et al., 2012). Second, growth kinetics and clonogenic post-cryopreservation capacity evaluation indicated that these cells were capable of proliferating without major variations between P₂-P₆; other authors have described the culture of bovine MSCs over a prolonged period with similar kinetics (LU et al., 2014a; SAMPAIO et al., 2015). Third, clonogenic capacity was higher in fresh than post-cryopreservation cells. There are few literature reports on the effect of cryopreservation on colonic or proliferative capacity and they do not describe important changes (DE MORAES et al., 2016).

In the present study, post-mortem Vs AD-MSCs were isolated, characterized and cryopreserved. Previous studies describe bovine AD-MSCs from different anatomic sites, such as the base of the tail (SAMPAIO et al., 2015, 2009), interdigital fat pad of the hoof (ZHAO et al., 2012), and abdominal fat (LU et al., 2014a). However, currently, there are no comparative studies on how the anatomic site can affect either the in vitro proliferative or multipotential capacity of AD-MSCs from cattle. By contrast, studies on species such as dogs (HENDAWY et al., 2021), humans (BAGLIONI et al., 2012) and rabbits (PEPTAN et al., 2006) described differences between harvesting sites.

We agreed with the researches of other investigators that isolation of post-mortem bovine adipose tissue (ZHAO et al., 2012) may be a convenient strategy because samples can be obtained from commercial abattoirs. However, in the present study, samples were taken from cow carcasses rather than the interdigital fat pad of the hoof.

Our MSC isolation success rate was 58.8 %, while other studies with cattle obtained 77% from umbilical cord blood (RAOUFI et al., 2011). However, the MSC isolation success rate is seldom reported (LU et al., 2014a; SAMPAIO et al., 2015; ZHAO et al., 2012). Moreover, bovine AD-MSCs post-cryopreserved for at least 1 year were used for proliferation capacity in successive passage and multipotency assays. In another study, MSCs retained their post-freezing characteristics (DE MORAES et al., 2016), and no significant change in proliferation capacity, multi-differentiation and chromosomal stability was reported in bovine endometrial MSCs. By contrast, in our study, only the colony-forming capacity of fresh and cryopreserved cells was compared, with greater capacity found in the former.

When proliferation capacity was evaluated through DT in successive passages, there were no significant variations for the subculture analyzed. The antecedents of bovine AD-MSCs are scarce; ZHAO et al. (2012) found no differences in DT until P₅, with a range of 16 to 22 h. This differs from other authors who reported 30 ± 1.9 h between P₁-P₉ (SAMPAIO et al., 2015), and from our results, which were greater than 24 h in all passages. One of the reasons for this short period could be the GM used, with the addition of Ham’s F-12 nutrient mixture. Another difference is that the authors used fresh cells (ZHAO et al., 2012) instead of their post-freezing counterparts, as used in this study. Finally, the anatomical site of cell extraction was also different, which could affect growth kinetics.

We obtained a mean of 79 h for DT between P₂-P₆, greater than the 30 h for P₁-P₉ described by SAMPAIO et al. (2015). This discrepancy could be because sampling was carried out from Sc fat, instead of Vs. In other species, differences in growth kinetics were observed, such as Sc AD-MSCs, which had a greater proliferative capacity (BAGLIONI et al., 2012; HENDAWY et al., 2021; PEPTAN et al., 2006). Culture conditions were different and their incubation temperature of 38.5 °C was higher than ours. They used α-MEM supplemented with 15 % FBS as GM, unlike our conditions. Additionally, the increase of FBS in the culture medium accelerates MSC growth kinetics (RUSSELL & KOCH, 2016).

Our DT results showed no significant changes across the passages, corroborating other authors who reported that bovine AD-MSCs could be used in this subculture range without losing their proliferative capacity (LU et al., 2014a; SAMPAIO et al., 2015; ZHAO et al., 2012). However, the heterogeneity of adipose tissue-harvesting sites, GM, and incubation conditions make it difficult to compare the DT of bovine cells. Furthermore, clonogenic capacity was evaluated by the CFU-F assay, with no significant variations between successive post-cryopreservation passages. This indicated that cells conserve their colony-forming capacity in culture, as other authors have described for this range of passages (LU et al., 2014a). Although, the literature indicates that colony-forming rates decrease as the number of passages increases, bovine MSCs retain their long-term colony-forming capacity even at > P₂₀ with cells from sources such as adipose tissue (LU et al., 2014a), bone marrow (LU et al., 2014b) and umbilical cord (XIONG et al., 2014).

Our results showed that the cells exhibited in vitro trilineage differentiation capacity (adipogenic, chondrogenic and osteogenic cell lineages), known lineages for MSC characterization (DOMINICI et al., 2006). We agreed with other authors who have achieved complete trilineage differentiation in bovine MSCs (SAMPAIO et al., 2015; ZHAO et al., 2018; ZHAO et al., 2012). Conversely, other studies characterized bovine MSCs but do not indicate the three mesodermal lineages (DE MORAES et al., 2016; LARA et al., 2017; LU et al., 2014a; LU et al., 2014b). However, some of these studies describe in vitro differentiation to other cell lineages such as hepatocytes, pancreatic islet cells and neurocytes (LU et al., 2014b; XIONG et al., 2014). Thus, it seems important to characterize bovine MSCs by demonstrating trilineage differentiation capacity, since the use of specific surface markers for bovine MSCs has yet to be established, as it has for humans (BOURIN et al., 2013; DOMINICI et al., 2006). Despite this situation for domestic animal MSCs (RANERA et al., 2011; SCREVEN et al., 2014), some studies have immunophenotyped bovine AD-MSCs (LU et al., 2014a; SAMPAIO et al., 2015).

CONCLUSION

The isolation, characterization, and cryopreservation of bovine AD-MSCs from post-mortem Vs fat was feasible, confirmed by fibroblastic morphology, adherence to plastic and in vitro trilineage differentiation capacity. Furthermore, these cells were cultured under standard conditions and successive cell passages up to P₆ showed proliferative capacity. Clonogenic capacity was higher in fresh than post-cryopreserved cells. Thus, post-mortem sampling of cattle carcasses for MSC isolation appears to be a promising source and future studies should be conducted to explore its applicability in cell therapy and the food industry.

ACKNOWLEDGEMENTS

The authors thank Universidad de la República (Udelar) for their financial support. The authors are grateful to Dr. Kent Erickson for his contribution to reviewing & editing.

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• CR-2023-0447.R1

BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL

• This study was carried out in strict accordance with the recommendations of the Honorary Commission for Animal Experimentation (CHEA) of Uruguay under approval identification CEUA-FVET no. 685.

Edited by

Editor: Rudi Weiblen (0000-0002-1737-9817)

Publication Dates

• Publication in this collection
  21 June 2024
• Date of issue
  2024

History

• Received
  17 Aug 2023
• Accepted
  15 Jan 2024
• Reviewed
  02 May 2024