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. 2014 Oct;78(4):290–296.

Application of a novel sorting system for equine mesenchymal stem cells (MSCs)

Catherine L Radtke 1,✉, Rodolfo Nino-Fong 1, Blanca P Esparza Gonzalez 1, Laurie A McDuffee 1
PMCID: PMC4170768  PMID: 25355998

Abstract

The objective of this study was to validate non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs) into subpopulations, for use with MSCs derived from equine muscle tissue, periosteal tissue, bone marrow, and adipose tissue. Cells were collected from 6 young, adult horses, postmortem. Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and left supragluteal subcutaneous adipose tissue. Aliquots of 800 × 103 MSCs from each tissue source were separated and injected into a ribbon-like capillary device by continuous flow (GrFFF proprietary system). Cells were sorted into 6 fractions and absorbencies [optical density (OD)] were read. Six fractions from each of the 6 aliquots were then combined to provide pooled fractions that had adequate cell numbers to seed at equal concentrations into assays. Equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells were consistently sorted into 6 fractions that remained viable for use in further assays. Fraction 1 had more cuboidal morphology in culture when compared to the other fractions. Statistical analysis of the fraction absorbencies (OD) revealed a P-value of < 0.05 when fractions 2 and 3 were compared to fractions 1, 4, 5, and 6. It was concluded that non-equilibrium GrFFF is a valid method for sorting equine muscle tissue-derived, periosteal tissue-derived, bone marrow-derived, and adipose tissue-derived mesenchymal stem cells into subpopulations that remain viable, thus securing its potential for use in equine stem cell applications and veterinary medicine.

Introduction

The need for less expensive, more practical, and more “cell-friendly” methods for cell sorting is an important next step in both equine and human cellular-based therapy (1,2). Fluorescence-activated cell sorting (FACS) is now the standard for sorting populations and subpopulations of cells (3). Cells can be sorted one at a time based on the specific light-scattering and fluorescent characteristics of each cell. While this method provides a quantitative reading of fluorescence from individual cells as well as allowing physical separation of cells of particular interest, the cells are tagged by antibodies and fluorescent labels in preparation for FACS, which changes their cell surface characteristics and impedes post-FACS assays (4). The vast expense and technical difficulty of a FACS system, the dedicated technical support required for its operation, and the fact that the reagents are prohibitive to many create a need for more attainable options for cell sorting.

The lack of a definitive consensus on equine mesenchymal stem cell (MSC) cluster designation (CD) marker expression is another complicating factor when using an immunological tagging system to sort equine stem cells (5). The markers are limited and often recognize multiple members of subpopulations of a stem cell lineage. As there is a lack of reactivity between commercial monoclonal antibodies and epitopes on equine cells, phenotyping has been incomplete (6). Current studies in the equine MSC field have shown positive expression of cell surface markers CD90 and CD44 (7,8) and negative expression of CD34 and CD45 (9). The presence of surface markers does not mean, however, that MSCs are in a completely undifferentiated state (4). Sorting the MSCs with a tag-less method would circumvent these issues with CD markers that plague equine MSC researchers.

Mesenchymal stem cells (MSCs) are available for isolation from many different tissues in horses. As they are sparse in numbers in post-natal tissues compared to embryonic tissues, sorting methods are required to separate MSCs from differentiated cells in the tissue. Pluripotent MSC subpopulations have also been identified in humans (9–12) and in rats (13). These subpopulations have been found to have different shapes, proliferation, and differentiation abilities (13,14). It is therefore important to be able to isolate the fraction of MSCs that proliferates and differentiates optimally for the application of interest.

Field-flow fractionation (FFF) is a group of bioanalysis techniques that have applications in the separation of bioanalytes ranging from proteins and nucleic acids to viruses, organelles, and whole cells. Gravitational field-flow fractionation (GrFFF) is a type of FFF technique that relies on the earth’s gravity to achieve sedimentation. Cells differing in molar mass, size, and surface antigens are driven by gravity into different velocity regions. The cells are then carried downstream through the channel at different speeds and exit the channel after different retention times. The distribution of the cells into the various resulting fractions reveals the separation characteristics (15). In traditional GrFFF systems, after a sedimentation step, equilibrium is reached between the gravitational field and the hydrodynamic forces of the transport fluid stream within the capillary channel. Cell sedimentation in the system tends to cause cells to adhere to the wall of the capillary channel and cell-cell aggregation/stacking. Due to the adherent nature of multipotent MSCs, non-equilibrium, earth gravity-assisted dynamic fractionation (NEEGA-DF), which skips the sedimentation step, is used to circumvent these MSC tendencies and to increase cell recovery (3,16).

It has been shown that GrFFF-based methods are of interest for cellular applications. The separation of neoplastic B-cells from healthy B- and T-cells in a heterogeneous blood sample has been described in a recent study (17). Gravitational field-flow fractionation (GrFFF) has been used to sort different human stem cells (18) and non-equilibrium GrFFF has been implemented to isolate, purify, and sort human MSCs from clinical specimens derived from different sources (16). Once separated from differentiated cells, the differences in the source of donor tissue MSC can be distinguished by the different elution profiles. Resulting fractions will have varied commitment potentials that correspond to their differing levels of stemness (4).

Our purpose in this study was to validate the use of non-equilibrium GrFFF as a sorting technique for: i) equine muscle-derived mesenchymal stem cells (MMSCs); ii) periosteal tissue-derived mesenchymal stem cells (PMSCs); iii) bone marrow-derived mesenchymal stem cells (BMSCs); and iv) adipose tissue-derived mesenchymal stem cells (AMSCs).

Materials and methods

Samples

Six young, adult horses (2 to 5 y of age) were used for postmortem collection of bone marrow, periosteum, skeletal muscle, and adipose tissue. The horses were donated to the Atlantic Veterinary College for reasons other than this study and were euthanized in accordance with protocols approved by the University of Prince Edward Island Animal Care Committee (2). All horses were first sedated with xylazine (Xylamax; Bimeda, Cambridge, Ontario), 1.1 mg/kg body weight (BW), IV and then euthanized with pentobarbital sodium injection (Euthanyl Forte; Bimeda), 10 mL/50 kg BW, IV.

Tissue collection and cell isolation

Techniques were carried out as described in an earlier study (2). Briefly, immediately after euthanasia, bone marrow (Illinois bone marrow biopsy needle; Carefusion, San Diego, California, USA) was aseptically collected from the sternebrae, adipose tissue (24 cm3) from the left subcutaneous supragluteal area lateral to the tail head, muscle (9 cm3) from the left semitendinosus/membranosus muscles, and periosteum (4 cm2) from the proximal medial surface of the right tibia. The aspirate (9.5 mL) of bone marrow was collected from the fourth sternebra into a 12-mL syringe that had been pre-loaded with 2.5 mL of 1000 IU/mL heparin (Leo Pharma, Thornhill, Ontario). Another sample was immediately drawn from the fifth sternebra in the same fashion and transported to the laboratory. Cells were isolated from bone marrow by a centrifugation gradient technique. The tissues collected were placed in alpha minimal essential media (MEM; Invitrogen, Toronto, Ontario) and transported to the laboratory. Cells were isolated from fat, muscle, and periosteum, all by means of the same enzyme digestion technique (2).

Cell cryopreservation

Cells were divided into 2.5 × 106 cell aliquots with 1.8 mL of freezing media [10 mL demethyl sulfoxide (DMSO) in 90 mL fetal bovine serum (FBS)] in cryo-vials (Corning, Corning, New York, USA). They were kept at −80°C for a minimum of 24 h and then placed in a liquid nitrogen tank until removed for cell culture. Viable cells were plated in T-75 flasks (Corning) at a cell density of 33 × 103 cells/cm2 in standard media [(MEM supplemented with 10% FBS (PAA Laboratories Etobicoke, Ontario), L-glutamine (2 mM) (Invitrogen, Toronto, Ontario), 10 000 U penicillin, 10 mg streptomycin/mL (Invitrogen), and 250 μg/mL amphotericin B (Invitrogen)].

Cell preparation

Cultured and expanded cells from passage 2 of each of the 4 sources of donor tissue (muscle, periosteum, bone marrow, and adipose) from 4 to 6 horses were used for the GrFFF. Periosteal-derived mesenchymal stem cells (PMSCs) and AMSCs were sorted from 4 horses, while MMSCs and MBSCs were sorted from 6 horses. Cells were washed with PBS (Invitrogen) and then incubated for 30 min in a humidified 5% carbon dioxide and 95% air atmosphere incubator at 37°C with 5 parts Versene (Invitrogen) to 1 part Tripson (Invitrogen). The reaction was stopped with an equal amount of standard media. The cell suspension was spun at 377 × g for 10 min and the supernatant removed. The pellet was vortexed and resuspended in 3 mL mobile phase solution [1 g BSA (bovine serum albumin; Fisher Scientific, Fair Lawn, New Jersey, USA) in 1 L PBS made with ultrapure water and 5000 U penicillin, 5 mg streptomycin/mL]. Aliquots of 800 × 103 cells from each sample were seeded into 6 Eppendorf vials, spun down at 377 × g for 7 min, and the supernatant was removed. Fifty microliters of mobile phase solution was added to each Eppendorf vial and cells were resuspended.

GrFFF System

The GrFFF system was purchased from byFlow (Bologna, Italy) and assembled and operated as per manufacturer’s instructions. The fractionation system and the 100-μL high-performance liquid chromatography (HPLC) syringe (Hamilton, Reno, Nevada, USA) to be used for sample loading were sterilized at the beginning of each working day as described and schematized in previous studies (3,18). Each aliquot of 800 × 103 cells was then individually injected into the GrFFF system (3,16) and sorted into 6 fractions by changing the collection tube every 5 min. This timing was based on human MSC sorting work done with the GrFFF system (3) and validation work done in our laboratory, which graphed the absorbency readings at different intervals and adjusted the collection times until fraction absorbencies were consistently different from 1 group to the next. The absorbencies (OD) of the 6 fractions were then characterized by spectrophotometric analysis (LKB Biochrom Ultrospec II 4050 UV/Vis Spectrophotometer; Biochrom, Holliston, Massachusetts, USA) at a wavelength of 600 nm. The 6 fractions from each of the 6 aliquots were combined to provide pooled fractions that were assessed by hemocytometric analysis and seeded at equal concentrations into assays as shown in Figure 1.

Figure 1.

Figure 1

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Steps of gravitational field-flow fractionation (GrFFF) cell sorting study design. Four tissues (bone marrow, adipose, muscle, and periosteum) were collected from 6 horses and mesenchymal stem cells (MSCs) cultured from each tissue. Each aliquot of 800 × 103 cells was then individually sorted by the GrFFF system into 6 fractions. The absorbencies [optical density (OD)] of each fraction were determined. The sorting process was done on 6 different aliquots and then resulting fractions were combined to provide pooled fractions for further culture.

Characterization of sorted MSCs

Sorted cells from each tissue source were seeded at a density of 1300 cells/cm2 into 30-mm dishes (Corning) and supplemented with and maintained for 1 wk in standard medium, after which adherence to the flask and spindle-shaped morphology was confirmed using direct microscopic analysis.

Cell differentiation

Cells from each tissue source from each of 3 individuals were induced to differentiate into the following 3 groups for evaluation: i) adipocyte; ii) chondrocyte; and iii) osteoblast. Each of the 3 lineages was cultured in parallel with 1 in standard media, as previously described. Light microscopy (Axiovert 40 CFL; Carl Zeiss Canada, Toronto, Ontario) digital images (PowerShot G5; Canon, Mississauga, Ontario) were taken on day 10 to assess the different morphologies. Histochemistry and morphology were used to confirm differentiation into the 3 lineages.

Adipogenic differentiation

Cells were seeded at a density of 1300 cells/cm2 into 35-mm wells. Cells were first cultured for 3 d in standard medium. Thereafter, the cells were exposed to an adipogenic induction medium (AM) [(DMEM/F12; Invitrogen), 3% FBS, 10 000 U penicillin and 10 mg streptomycin/mL, 250 μg/mL amphotericin B, 33 μmol/L biotin (Sigma), 17 μmol/L pantothenate (Sigma), 1 μmol/L insulin (Sigma), 1 μmol/L dexamethasone (Sigma), 0.5 mmol/L isobutylmethylxanthine (IBMX; Sigma), 5 μmol/L rosiglitazone (Toronto Research Chemicals, Toronto, Ontario), and 5% rabbit serum (Invitrogen)] for 2 d. Thereafter, the same medium without the IBMX and the rosiglitazone was used to maintain the adipocyte cell culture until day 7 when the cells were fixed for 20 min in 10% neutral buffered formalin (Fisher Scientific, Nepean, Ontario) at room temperature and stained for neutral lipid accumulation with Oil Red O, indicating adipogenic differentiation (19).

Chondrogenic differentiation

Cells were seeded at a density of 1300 cells/cm2 into 35-mm wells and supplemented with a chondrogenic differentiation medium (CM) [(Hams 12; Sigma), dexamethasone (10-7 M), ITS+1 (Sigma) (culture supplement containing bovine insulin, transferrin, selenous acid, linoleic acid, and BSA) 5% FCS, 10 000 U penicillin and 10 mg streptomycin/mL, 250 μg/mL amphotericin B, 50 μg/mL ascorbic acid (Sigma), and 1 ng/mL recombinant human transforming growth factor-beta 1(rhTGF-beta1) (Millipore, Temecula, California, USA)]. Cultures were maintained for 7 d and then fixed for 20 min in 10% neutral buffered formalin at room temperature. Cultures were then stained with Alcian blue pH 1.0 for the detection of sulfated proteoglycans to confirm chondrogenic differentiation.

Osteoblastic differentiation

Cells were seeded at a density of 1300 cells/cm2 into 35-mm wells. Cells were supplemented with an osteogenic induction medium (OM) [(α-MEM, 5% FCS, 10 000 U penicillin and 10 mg streptomycin/mL, 250 μg/mL amphotericin B, 50 μg/mL ascorbic acid, dexamethazone 10−8, and 10 mM B-glycerophosphate (Sigma)]. Cultures were maintained for 7 d and then fixed for 20 min in 10% neutral buffered formalin at room temperature. Cultures were then stained for calcium with von Kossa stain (20) and with the substrate naphthol AS MX-PO4 and Red Violet LB salt for alkaline phosphatase (21) to confirm mineralization and osteoblastic differentiation.

Statistical analysis

Fraction absorbancies were compared using a paired t-test with horse as the experimental unit. Significance was set at P < 0.05.

Results

Spectrophotometric analysis of each fraction

Muscle-derived mesenchymal stem cells (MMSCs), PMSCs, BMSCs, and AMSCs were sorted by GrFFF into 6 fractions, which was repeated 6 times. The fraction contents were compared by spectrophotometric absorbance (Figures 2a–d) as an objective comparison of cell number and size. The OD values of fractions 1 and 5 are very close to zero (readings in thousandths) due to the small size and number of the cells and fraction 6 was zero due to a complete lack of cells. Absorbencies of fraction 2 and 3 compared to fractions 1, 4, 5, and 6 for each tissue revealed a significant difference (P < 0.5). In general, fractions with higher absorbencies also had higher cell counts and fractions with lower absorbencies had lower cell counts.

Figure 2a.

Figure 2a

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Graph of absorbance [optical density (OD)] versus fraction for 6 horses (bold numbered panels) and replicates 1 to 6 (lines) of sorted cells derived from muscle tissue. Note the consistency in absorbency between fraction replicates. Fraction 1 (circles), fraction 2 (squares), fraction 3 (diamonds), fraction 4 (triangles), fraction 5 (right arrowhead), and fraction 6 (left arrowhead).

Figure 2d.

Figure 2d

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Graph of absorbance [optical density (OD)] versus fraction for 4 horses (bold numbered panels) and replicates 1 to 6 (lines) of sorted cells derived from adipose tissue. Note the consistency in absorbency between fraction replicates. Fraction 1 (circles), fraction 2 (squares), fraction 3 (diamonds), fraction 4 (triangles), fraction 5 (right arrowhead), annd fraction 6 (left arrowhead).

Microscopic analysis of each fraction

Each type of mesenchymal stem cell (MMSC, PMSC, BMSC, and AMSC) from each horse had cells in fractions 1 to 5, but none in fraction 6. Cells from all cell sources and fractions adhered to plastic. The cell recovery from the GrFFF sorting system was poor overall and ranged from 28% to 73%. This range was attributed to inter-horse variation. The highest number of cells was found in fractions 2, 3, and 4 in all tissues. The morphology of cells in fraction 1 was more cuboidal, while cells in fractions 2 to 5 were more classic, fibroblastic spindle shapes, which indicates that different subpopulations were indeed separated from one another (Figure 3). Muscle-derived mesenchymal stem cells (MMSCs), PMSCs, BMSCs, and AMSCs were sorted by non-equilibrium GrFFF, while maintaining sterility and viability.

Figure 3.

Figure 3

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Representative photomicrographs of differing morphology between fractions of mesenchymal stem cells (MSCs) cultured from equine muscle sorted by gravitational field-flow fractionation (GrFFF). Note that the morphology of cells in fraction 1 was more cuboidal than in fractions 2 to 5, which were more classic, fibroblastic spindle shapes. All are unstained. Scale bar — 200 μm.

Trilineage differentiation

Cells from fractions 1 to 5 from each tissue were able to undergo trilineage differentiation as seen in the representative photomicrographs in Figure 4. Cells cultured in adipogenic differentiation medium for 4 d had positive results for Oil Red O staining of lipid droplets. Cells cultured in standard medium did not develop lipid droplets and lacked staining with Oil Red O. Mesenchymal stem cells (MSCs) that were cultured in chondrogenic differentiation medium for 7 d stained positively for glycosaminoglycans with Alcian blue. Cells cultured in standard medium lacked Alcian blue stain uptake. Cells cultured in osteogenic differentiation medium for 7 to 10 d formed bone nodules based on positive results of alkaline phosphatase and calcium-specific stains. Cells cultured in standard medium did not develop nodules and lacked staining for alkaline phosphatase and calcium.

Figure 4.

Figure 4

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Representative photomicrographs of GrFFF sorted MSCs (fractions 1–5 from each tissue) after tri-lineage differentiation and histochemical staining. Osteogenic medium is stained with von Kossa stain (A), adipogenic medium is stained with oil red O (B), chondrogenic medium is stained with Alcian blue (C), and standard medium is unstained (D). Scale bar is 200 μm.

Discussion

This is the first study to validate the use of non-equilibrium GrFFF as a sorting method for equine-derived MMSCs, PMSCs, BMSCs, and AMSCs. The cells from each source were driven by gravity into different velocity regions and successfully sorted into fractions, which showed that they possess differences in molar mass, size, or surface antigens. The fractions with higher absorbencies also had higher cell counts, while those with lower absorbencies had lower cell counts. Cell size likely also plays a part in the absorbency measurements as larger cells travel more quickly through the chamber than smaller cells.

Muscle-derived mesenchymal stem cells (MMSCs), PMSCs, BMSCs, and AMSCs used for non-equilibrium GrFFF sorting in this study were characterized by morphology, adherence to plastic, trilineage differentiation, and stem cell surface marker detection by immunofluorescence and flow cytometric analysis for a previous study (2). Although populations of cells from each tissue had a high percentage of purity based on the flow cytometry results from this previous work, it became obvious in the present study that there were several subpopulations in each sample. As the populations sorted into fractions, each of which had different absorbencies, it was concluded that they were from different subpopulations. These subpopulations were found to have trilineage differentiation capabilities and were therefore not altered by the sorting process. Evidence from this study indicates that cell sorting by properties other than cell surface markers is essential when the phenotype has not been completely elucidated.

Many aspects of the non-equilibrium GrFFF technique make it ideal for use in cell culture. Non-equilibrium GrFFF allows MSC isolation time to be improved by clearing other cells and contaminates from the sample in an early passage. Conventionally, this is achieved by adherence and detachment cycles implemented over several passages during cell culture. The shortening of this cleanup phase with a technique such as GrFFF is of great benefit in clinical application of stem cell therapy as it shortens the return time on samples received for injured patients (3,22).

The GrFFF technology is similar to fluorescence-activated cell sorting (FACS) in that it is a system to sort subpopulations of cells, but it is superior in several aspects. The non-equilibrium GrFFF is a tag-less system of stem cell sorting that will avoid augmentation of the MSCs (3). The GrFFF sorting system is also very economical as it can be assembled and maintained in the laboratory from inexpensive instruments and reagents owned by most biotechnology laboratories or that can be purchased for a fraction of the cost of a FACS machine. The GrFFF system is also far less technically difficult to operate than the FACS system. Perhaps most importantly, GrFFF allows the MSCs to be maintained under sterile conditions, which also allows for further culture, expansion, assays, and use in cell-based therapies after fractionation (4). In this study it was also confirmed that MMSCs, PMSCs, BMSCs, and AMSCs can be sorted by non-equilibrium GrFFF while maintaining viability for further assays.

The main limitation of the GrFFF system is the low number of cells that can be fractionated per run. Another study discovered a polar hydrophobic environment on the polyvinyl chloride (PVC) material used in the GrFFF system, which explains the low recovery of biological samples. The sample returns improved when a coating was placed on the PVC (23). As we were validating the system for use with equine MSC for the first time, we choose to use the system in its simplest form. We increased sorting throughput by pooling fractions collected at the same retention times from repeated runs. After validation of a single system with human lymphocytes, other researchers have set up 2 GrFFF channels in parallel to increase sorting throughput (18).

In equine regenerative medicine, identifying the optimum source of MSCs, or other progenitor cells, for each application has been the focus of much evaluation (2,24–26). This research points out that the variation of MSCs within each source must also be taken into consideration. Techniques for sorting and enrichment of MSCs may be the key to isolation of the equine MSC phenotypes. To this end, future goals include using non-equilibrium GrFFF for purification and fractionation of MSCs into subpopulations to be evaluated for CD markers using flow cytometry. Once the phenotype is elucidated, comparative assays between the fractions will determine the optimum source and CD markers of MSCs for the intended application. With this information, the GrFFF system can be used to isolate the subpopulation of interest, which will allow culture and expansion of an ultrapure population.

In conclusion, the ability to affordably and effectively sort equine mesenchymal stem cells (MSCs) with non-equilibrium GrFFF broadens the choices available to clinicians using MSCs in cell-based therapies.

Figure 2b.

Figure 2b

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Graph of absorbance [optical density (OD)] versus fraction for 4 horses (bold numbered panels) and replicates 1 to 6 (lines) of sorted cells derived from periosteal tissue. Note the consistency in absorbency between fraction replicates. Fraction 1 (circles), fraction 2 (squares), fraction 3 (diamonds), fraction 4 (triangles), fraction 5 (right arrowhead), and fraction 6 (left arrowhead).

Figure 2c.

Figure 2c

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Graph of absorbance [optical density (OD)] versus fraction for 6 horses (bold numbered panels) and replicates (lines 1 to 6) of sorted cells derived from bone marrow. Note the consistency in absorbency between fraction replicates. Fraction 1 (circles), fraction 2 (squares), fraction 3 (diamonds), fraction 4 (triangles), fraction 5 (right arrowhead), and fraction 6 (left arrowhead).

Acknowledgments

The authors thank Glenda Wright, PhD for her assistance with this manuscript. This study was supported by the Atlantic Canada Opportunities Agency and Innovation PEI.

Footnotes

This manuscript represents a portion of a thesis to be submitted by Dr. Radtke to the University of Prince Edward Island, Department of Health Management, as a partial fulfillment of the requirements for a Doctor of Philosophy degree.

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