Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

Thokchom Shitarjit Singh1, O.R. Sathyamoorthy2, Soundian Eswari3,*, Sabiha Hayath Basha4, M. Parthiban5
1Department of Veterinary Anatomy, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
2Department of Veterinary Anatomy, Veterinary College and Research Institute, TANUVAS, Theni-625 602, Tamil Nadu, India.
3Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, TANUVAS, Chennai-600 007, Tamil Nadu, India.
4Department of Veterinary Anatomy, Veterinary College and Research Institute, TANUVAS, Salem-636 112, Tamil Nadu, India.
5Department of Animal Biotechnology, Madras Veterinary College, TANUVAS, Chennai-600 007, Tamil Nadu, India.
Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells.

Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated.

Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.
Stem cell biology is one of the most vital areas in biomedical research today (Gimble et al., 2007). Mesenchymal stem cells (MSCs), also called as mesenchymal stromal cells or stromal stem cells, reside in the stroma of most organs (Wei et al., 2013). It offers a great deal of excitement and promise for development of cell-based therapeutic strategies, primarily owing to their intrinsic ability to self-renew and differentiation properties. MSCs are considered as a readily accepted source of stem cells because such cells have already been demonstrated efficacy in multiple types of cellular therapeutic strategies in humans, including applications in treating with hematopoietic recovery (Koc et al., 2000), osteogenesis imperfecta (Horwitz et al., 2002) and bone tissue regeneration strategies (Petite et al., 2000). Adipose tissue, as a stem cell source is available extensively and has several advantages as compared to other sources. It is easily accessible in large quantities with minimal morbidity upon harvest. Adipose derived mesenchymal stem cells (AD-MSCs) shows potential for multiple differentiation (Cowan et al., 2004). Culture for AD-MSCs is simple to generate because of their higher intrinsic proliferative rate and maintenance of their phenotypic characteristics (Bunnell et al., 2008). MSCs are identified using three characteristics: (1) adherence to the flask bottom (2) characterized the immunoprofile of heterogeneous polyclonal population of undifferentiated cells using different MSC/ADSC markers such as CD44, CD90,  CD105 (Dominici et al., 2006), CD9, CD10, CD13, CD29, CD54, CD55, CD71,  CD91, CD34, CD49d and CD106 (Mauneya et al., 2007). Hence, the  present experiment is aimed to isolate, characterize and to study the morphology of porcine AD-MSCs under good manufacturing practice (GMP).
In vitro culture and expansion of pAD-MSCs
Approximately 10 g of subcutaneous fat were harvested from the buccal area from Large White Yorkshire pigs (n=6) between the age of 6-8 months from Department of Livestock Products Technology, Madras Veterinary College, Chennai. Samples were transported on ice in Dulbecco’s phosphate-buffered saline (dPBS, Gibco®) containing 200 U/ml of Penicillin and 200 g/ml of Streptomycin and 2.5 g/ml of Fungizone to the GMP facility of Centre for Stem Cell Research and Regenerative Medicine . The tissue was finely minced into fragments of 4-5 mm and washed two to three times with dPBS containing antibiotics and antimycotics (ABAM, Gibco®). Tissue samples were homogenized with collagenase type II powder (Sigma Aldrich, USA, 900 units of collagenase/1.5 ml DMEM/g fat) in incubator at 37°C for 2 hours by shaking the sample at an interval of every 20 minutes.

After digestion the cell suspension was filtered through 100 μm nylon filter system into 50 ml conical centrifuge tubes, centrifuged at 1200 rpm for 10 minutes to collect the pellet of stromal-vascular cells. The supernatant was decanted and 10 ml of dPBS was added into each tube to resuspend the pellet by repeated pipetting and centrifuged at 1200 rpm for 6 minutes twice. The cells were suspended into 5 ml Dulbecco’s modified Eagle medium (DMEM, Gibco®) through 70 μm filter system. The cells were counted with a hemocytometer (Neubauer’s chamber) with a aliquot 20 μl of cell-containing medium mixed with 20 μl of 0.4% trypan blue solution (1:1 dilution) (Neupane et al., 2008). The isolated cells were plated at 1.5 x 106 cells per T25 culture flask in DMEM with high glucose supplemented with 10% fetal bovine serum (FBS, Gibco®), 100U/ml of Penicillin and 100 μg/ ml of Streptomycin and 2.5 μg /ml of Fungizone and incubated at 37°C in 5% CO2.

The culture medium was replaced with fresh medium in every 3-4 days. At 70-90% confluence, adipose derived stem cells were detached, subcultured with 0.25% Trypsin-EDTA solution (Sigma®), re-seeded at an initial concentration of 1.5 x 105 cells per T25 culture flask and maintained upto passage 5.
Proliferation assays
The cell doubling (CD) and population doubling time (PDT) were determined with replicate cultures of P2–P3 cells according to standard methods (Rutigliano et al., 2006).
CD= In (Nf/Ni)/In (2)
CT= Culture time, Nf- Final cell number, Ni- Initial seeding density, In- Natural logarithm.
RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR)
The messenger RNA (mRNA) from undifferentiated cells were isolated at passage 3 using a Qiagen RNeasy kit (Qiagen, USA) and complementary deoxyribonucleic acid (cDNA) was synthesized using iScript ®(Bio-Rad Laboratories, USA) cDNA Synthesis Kit. PCR was performed with primers for MSC specific marker genes CD 44, CD73, endogenous control β-actin and negative marker CD 34 (Table 1). The PCR program used for the amplification of all genes consisted of the following: a denaturing cycle of 5 min at 95°C, 35 cycles of PCR (95°C for 30 s, 54°C to 58°C for 30 s and 72°C for 60 s) and a step cycle starting at 72°C for 10 min. PCR products were subjected to electrophoresis using a 2% agarose gel, stained with ethidium bromide and illuminated with UV light.

Table 1: Primer sequences for RT-PCR.

Immunocytochemistry was carried out to localize the MSC specific marker proteins in the pAD-MSCs using specific primary antibody with its appropriate FITC conjugated secondary antibody. Cells grown in glass chamber slides were fixed with 4% paraformaldehyde for 20 min, washed in DPBS and the cells were then permeabilized with 0.2% Tween 20 and nonspecific binding was blocked with 5% bovine serum albumin by using BSA Blocking buffer for 1 h then washed with DPBS for three times. Then, cultures were incubated with 1:100 dilution of the rat primary antibody  for CD44 (ab119335; Abcam) and rabbit  primary antibody for CD34 (ab81289; Abcam) and allowed to act for overnight at 4°C. Primary antibody was then removed from wells and plates were washed with DPBS. After washing in DPBS, cell monolayers were incubated with FITC Conjugated  Goat Anti-Rat IgG (ab6840; Abcam) and Goat Anti-Rabbit IgG (ab6717; Abcam ), 1:1000 dilution for 1 h. Cells were washed in DPBS, stained with 40, 60-diamidino-2-phenylindole (DAPI) 1 µg/ml and mounted with Vectashield mounting medium. Specimens were examined under Inverted Phase Contrast Microscope (Nikon Eclipse Ti2).

Morphometry of pAD-MSCs
The cells were plated at 5 x 103 per well in 12 well plate in 20 μl culture medium onto glass coverslips for 60 minutes at 37°C and 5% CO2 to cellular adherence. After that, 500 μl of culture medium was added on each plate and maintained for 24 h and 48 h (Maciel et al., 2014). The culture medium was removed and the coverslips were stained with Leishman’s stain and mounted with DPX and observed under Inverted Phase Contrast Microscope (Nikon Eclipse Ti2).

Fifty cells at P2 and P3 were morphologically evaluated at 200X to measure length and width of the cells and their nuclei. One way ANOVA at 5% level was used between various passage levels. All analysis was done by using SPSS-16. Statistical calculations (mean ± standard error) were recorded according to the standard statistical procedures recommended (Snedecor and Cochran, 1994).
In vitro culture and expansion of pAD-MSCs
In this study, the average number of mononuclear cells was 2.0 x 106/10 g of each fat sample which was successfully established from 6 adult pigs (6-8 month age). The isolated cells were seeded at a density of 1 x 106 cells per T25 culture flask. Out of those mononuclear cells, an estimate of 25-26% adhered to the surface of culture flask and the rest are removed during the medium change after 24h of seeding. However, average mononuclear cells was  2.7 x 106/10g in pig (Williams et al., 2008), 5.5 × 104 ± 3.3 x 104 from subcutaneous interscapular adipose tissue (ScI-pASCs)/ml and 3.0 × 104 ± 9.3 x 103 from buccal fat pad (BFP-pASCs)/ml of raw pig tissue (Niada et al., 2013), 2-6 x 106 cells per 300 ml of human adipose tissue lipoaspirate (Zuk et al., 2001), 2.12±0.19 x 106/ ml of ovine subcutaneous fat (Gnanadevi et al., 2019). This difference in the number of cells may be due to difference in enzyme used, digestion time and species difference. By day 1, the adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology as in pig (Williams et al., 2008; Niada et al., 2013 and Liu et al., 2016) (Fig 1 and 3). 60-70% confluency was observed on day 4 to 5, cells become large and flatten with enlarge nucleus in the center of the colony and the colony united together forming a monolayer of pAD-MSCs in the culture flask. Cell culture expanded to 80-90 % confluence by 8-9 days after the initial seeding (Fig 2). This was similar to the observation in pig (Williams et al., 2008) and in ovine umbilical cord Wharton’s jelly (Eswari et al., 2016a and 2016b). However, full confluency was observed on 6th day in ovine (Grzesiak et al., 2011).

Fig 1: Photomicrograph of pAD-MSCs (a) showing plastic adherence of adhered cells (A) and non-adhered cells (NA) after 24 hour of primary culture (b) showing colony forming unit (C), spindle (S), round (R) and elongated (E) fibroblastic morphology adhered to plastic within 3 day of initial seeding. Scale bar=100 mm.

Fig 2: Photomicrograph of pAD-MSCs (a) showing 60-70 percent of confluency after 5 days (b) 90-100 per cent of confluency after 8 days at Passage 0 level (100X).

Fig 3: Undifferentiated stromal cell morphology showing (A) elongated shape (black arrow) and spindle shape cell with cytoplasm at one end of the cell (red arrow) (400X) (B) spindle shaped cells with single and two nucleolus (black arrow) and rounded widespread cells with abundant cytoplasm (red arrow) (400x) (C) cells demonstrating different spindle-like fibroblastic morphology adhered to plastic within 24h of Passage 2 (Leishman’s stain, 200x).

Cell culture maintenance
The in vitro cultured cells after 80-90% confluence were subjected to passage 1 by re-seeding at an initial concentration of 1.5 x 105 cells per T25 culture flask. Passage 1 cells showed 80-90% confluency after 3-4 days and were subjected to passage 2. In this study, pAD-MSCs were subcultered upto passage 5.
Proliferation assays
The proliferation rate of pAD-MSCs was calculated from P1 to P3 culture to access the cell doubling (CD) and population doubling time (PDT). At P1 and P2, cell doubling was found to be same 0.32 ×104 at 48 hours. The cell doubling at P3 was higher than P1 and P2 measured about 0.69 ×104 at 48 hours. Similarly at 96 h, 144 h and 192 h, the cell doubling of pAD-MSCs increases with increase in time. At P2 and P3 there was a final plateau phase at 96 hours. The population doubling time was found to be similar for P1 and P2 but decreases in P3 at 48 h. At 96 h, 144 h and 192 h, the doubling time of pAD-MSCs decreases with increase in passage level.

Thus when the cell doubling of pAD-MSCs increased, the population doubling time was decreased from P1 to P3. Lesser the population doubling time, greater was the proliferation rate. Hence P3 culture had a higher proliferation rate than P2 and P1. Growth curve was evaluated by counting the harvested cells at the end of each passage level from P1 culture to P3 and found to be increasing in all the passage (Fig 4 and Fig 5). However there were trends for cell doubling time to increase and cell doubling numbers to decrease for both adipose tissue-derived stromal cells and bone marrow-derived stromal cells with increasing cell passages in canine (Spencer et al., 2012). These changes in CD and PDT may be due to change with species variation, differences in the culture technique (Kamishina et al., 2008) and seeding density (Neuhuber et al., 2008) that affect the proliferation rate.

Fig 4: Cell doubling of porcine adipose derived mesenchymal stem cells at different passage (P1-Passage 1, P2-Passage 2 and P3-Passage 3).

Fig 5: Population doubling time of porcine adipose tissue derived mesenchymal stem cells at different passage (P1-Passage 1, P2-Passage 2 and P3-Passage 3).

Expression of MSC specific markers by RT-PCR
Gel electrophoresis analysis revealed the presence of both MSC specific markers CD44, CD73 and negative expression of CD34, the hematopoietic surface marker (Fig 6). Previous studies have defined that the undifferentiated human adipose tissue-derived stromal cells demonstrated positive staining for stem cell surface markers CD13, CD29, CD44, CD54, CD55, CD59, CD105, CD106, CD146 and CD166 and an absence of CD14, CD31, CD34 and CD45 expression (Gronthos et al., 2001). However, RT-PCR on pig adipose-derived progenitor cells indicated the expression for CD29, CD71, CD73, CD105 and CD166 but didn’t express for endothelial marker CD31 (Zhang et al., 2016).

Fig 6: RT PCR analysis of mesenchymal specific marker gene expression in pAD-MSCs. Lane1 - DNA marker (100 bp); Lane 2- CD44 (245 bp); Lane 3 - CD73 (411 bp); Lane 4 – CD34 negative marker; Lane 5 –â actin (223 bp).

Immunocytochemical localization of MSC specific proteins
Immunocytochemistry revealed that mesenchymal stem cells from porcine adipose tissue were positive for the MSC marker CD44 (Fig 7A) and negative for hematopoietic cell marker CD34 (Fig 7B). Similar report was present in goat adipose-derived stem cells (ADSCs) (Ren et al., 2012), in human ADSCs (Ghiasi et al., 2016) and in pig adipose-derived progenitor cells (Zhang et al., 2016). However, it is reported that expression of the MSCs specific markers were found to differ among passages, adipose tissue-derived mesenchymal stem cells (ATMSCs) at passage 0, expressed higher CD34 and CD45 and lower CD73, CD90 and CD105. So, with the increasing time of ATMSCs in culture, hematopoietic lineage markers (CD34, CD45) were decreased, while expression of CD73, CD90 and CD105 intensified (Yin et al., 2014).

Fig 7: Immunocytochemistry of pAD-MSCs. Photomicrographs show (A) Strong expression for CD44 MSCs specific marker (Green fluorescence) with DAPI (blue fluorescence) the nuclear stain (B) negative expression for CD34 the hematopoietic surface marker with DAPI (200X).

Cell morphometry of pAD-MSCs
The results of the statistical inference of the cells with regard to length and width of the pAD-MSCs and nucleus in P1 and P3 were showed in Table 2 and 3, respectively.

Table 2: Measurements (ìm) of pAD-MSCs passage 2 -24 and 48 hours of culture (200x).

Table 3: Measurements (ìm) of pAD-MSCs passage 3 -24 and 48 hours of culture (200x).

From the present study, the morphometric differences were reported for porcine adipose derived mesenchymal stem cells. At passage 2, cell length, nucleus length, cell width and nucleus width increased between 24 and 48 hours. Similarly at passage 3, cell length, nucleus length, cell width and nucleus width increased between 24 and 48 hours. However there was no significant difference noted in both length and width of cell and also length and width of nucleus 24 and 48 hours of culture in passage 2 and 3. Similar finding was reported in human MSCs (Docheva et al., 2008) when compared between 24 and 120 hours, in equine (Grzesiak et al., 2011) and in feline (Maciel et al., 2014) when compared at passage 1 and 3 in 24 and 120 hours.
The techniques used in this study can be used to increase the efficiency of isolation and purification of a population of mesenchymal stem cells from easily obtainable adipose tissue. The isolation of a somatic stem cell population from a model animal, such as pig can be used in tissue engineering and applications in regenerative medicine. The availability of such an animal model can be used in determining the true efficiency in comparing the somatic stem cell tissue with embryonic stem cells.
The author acknowledges the Professor and Head, Centre for Stem Cell Research and Regenerative Medicine (CSCR and RM) for permitting to utilize the GMP and other facilities available in CSCR and RM, Madras Veterinary College, Chennai to carry out this work.

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