Adipose Tissue Collection
Canine abdominal subcutaneous adipose was collected during routine clinically indicated surgery at Affiliated Animal Hospital of Foshan University from 2017 to 2019. All owners agreed with the collection of tissue and signed an informed consent. A screen of the medical history was performed and a blood sample was tested for specific canine pathogens, such as rabies virus (RV), canine distemper virus (CDV), infectious canine hepatitis virus (ICHV), canine parvovirus (CPV), canine coronaviruses (CCV), fungi and bacteria. Tissue collection was approved by the Animal Ethics Committee of Foshan University, and were conducted in accordance with the ethical standards of the university. Adipose tissue immersed in DMEM (Hyclone, USA) supplemented 1% penicillin and streptomycin (Gibco, USA) immediately transported to the lab at 4-10℃.
Cell Isolation and Culture
MSCs were isolated from adipose tissue based on methods previously described[40]. Briefly, tissue was digested with collagenase type I. Then filtration through a 100-mesh cell strain, the filtrate was centrifuged to collect AD-MSCs. Approximately 5000 isolated suspended cells per cm2 were transferred to cell culture flask (Corning, USA) in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel), 1% Pen-Strep (Gibco, USA), and 1% L-glutamine (Gibco, USA) and placed into the incubator at 37 °C in a humidified incubator containing 5% CO2. Cells from passage 2 were harvested during the first expansion period.
Cryopreservation
About 3×106 AD-MSCs at passage 2 were suspended in 1 ml of cryoprotectant solution containing 10% dimethyl sulfoxide (Me2SO)(Sigma-Aldrich, USA)and 90% FBS (Biological Industries, Israel).The cell suspension was kept in freezing tube (Corning, USA), cooled to −80 °C at a rate of −1 °C/min in freezing container (Nalgene, USA), and then transferred to nitrogen tank for long-term storage. At each freeze-thaw cycle, the stem cell characteristics of AD-MSCs were evaluated (Table 1).
Table 1. In-Process Test and Cryopreservation Criteria
Specification
|
Expected
|
Methods
|
Donor virology (RV, CDV, ICHV, CPV and CCV)
|
Negative
|
Elisa Kit
|
Viability
|
≥90%
|
Trypan blue staining
|
Sterility
|
No growth
|
Microbiology culture
|
Mycoplasma
|
Negative
|
Culture
|
Endotoxin
|
<0.5 EU/mL
|
Limulus Amebocyte Lysate
|
Phenotype (CD29, CD34, CD44, CD45 and CD90)
|
≥95% CD29, CD34, CD90
≤2% CD34, CD45
|
Flow cytometry
|
Differentiation potential
|
Osteogenesis, adipogenesis
|
Induction culture
|
Thawing and Expansion
AD-MSCs was taken from nitrogen tank and rapidly thawed in water bath kettle of 37°C and transferred to a 15 ml centrifuge tube in 10×volume of PBS for washing twice. The thawed cells were cultivated and proliferated using the same protocol as described for primary expansion. AD-MSCs from passage 3 to 7 were harvested for cytotherapy during this expansion period. All the cells for cytotherapy were evaluated synchronously (Table 2).
Table 2. Final Product Release Criteria
Specification
|
Expected
|
Methods
|
Cell counts
|
1.0×106 /kg
|
Automatic cell counter
|
Viability
|
≥90%
|
Trypan blue staining
|
Sterility
|
No growth
|
Microbiology culture
|
Mycoplasma
|
Negative
|
Culture
|
Endotoxin
|
<0.5 EU/mL
|
Limulus Amebocyte Lysate
|
Phenotype (CD29, CD34, CD44, CD45 and CD90)
|
≥95% CD29, CD34, CD90
≤2% CD34, CD45
|
Flow cytometry
|
Differentiation potential
|
Osteogenesis, adipogenesis
|
Induction culture
|
Karyotype analysis
|
39 pairs of chromosomes
|
Giemsa staining
|
Tumorigenicity
|
Negative
|
Injection into nude mice
|
Characterization and Quality Control
Cell Counts and Viability Assessment Accurate assessment of cell count and viability of AD-MSCs by the trypan blue dye exclusion test using automatic cell counter (Countess, Invitrogen, USA). The viability was calculated using the following formula: number of trypan blue-negative cells/number of total cell cells × 100.
Flow Cytometry Analysis Cells were incubated with the following phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated antibodies:anti-CD29-PE (cat.no.303004; BioLegend), anti-CD34-PE (cat.no.ab23830; Abcam), anti-CD44- FITC (cat.no.MA1-10229; Invitrogen), anti-CD45-FITC (cat.no.ab27287; Abcam), and anti-CD90-PE (cat.no.11-0900-81; Invitrogen) or their respective isotype controls. Cells were analyzed using a FACSCanto flow cytometry system (Beckman, USA). Data acquisition and analysis was performed with CytExpert.
In Vitro Differentiation Assessment In vitro adipogenic, osteogenic and chondrogenic differentiation were examined using MSCs Adipogenic Differentiation Kit (Cyanogen, China), Osteogenic Differentiation Kit and Chondrogenic Differentiation Kit (Cyanogen, China) following the manufacturer’s protocol for each kit. Adipogenic Differentiation Kit contains basic medium, fetal bovine serum, dexamethasone, insulin, IBMX and indomethacin. Osteogenic Differentiation Kit contains basic medium, fetal bovine serum, ascorbate, β-Glycerophosphate and dexamethasone. Chondrogenic Differentiation Kit contains basic medium, fetal bovine serum, ascorbate,dexamethasone,sodium Pyruvate, TGF-β3, insulin, transferrin and selenium. Cells were stained with Oil Red O solution to assess adipogenic differentiation, alizarin red solution to assess osteogenic differentiation and alcian blue solution to assess chondrogenic differentiation.
Population Doubling Time (Td) Estimation Cells were counted by automatic cell counter and cell population doubling time (Td) was calculated using the following formula: Td = t x lg2 / (lgNt - lgNo), where “No” refers to cell number after inoculation and “Nt” refers to cell number at T hour culture.
Colony-Forming unit-fibroblasts Estimation AD-MSCs were seeded in 60-mm Petri dishes (100 cells/dish) cultured for 10 days, and stained with crystal violet solution. The number of colonies was determined under a camera, and clusters of more than 50 cells were considered colonies. The Colony-Forming unit-fibroblasts(CFU-F)was calculated using the following formula: CFU-F = colony number / initial cell number x 100%.
Microbiology testing Mycoplasma, Bacterium and fungus examination were performed in accordance with methods set forth in the Chinese Veterinary Pharmacopoeia. Sterility test was performed by inoculating samples in two different sterile nutrient mediums, namely, Fluid Thioglycolate Medium and Soybean Casein Digest Medium. Mycoplasma detection was done by inoculating samples in Mycoplasma Agar Medium and Mycoplasma Broth Medium.
Endotoxin testing Endotoxin levels were determined by the gel clot limulus amebocyte lysate test in accordance with methods set forth in the Chinese Veterinary Pharmacopoeia.
Karyotype analysis Chromosomes were prepared and banded by the G-banding technique according to standard methods. Following Giemsa staining, the numbers of chromosomes were calculated and analyzed.
Tumorigenicity assay AD-MSCs and Hela (positive control) cells were delivered into the flank of NOD/SCID mice. After 40 days, mice were euthanized and tumors were excised. Tumors were fixed by immersion in neutral buffered formalin and processed for standard hematoxylin and eosin staining.
A Case of Cell Therapy
A 2-month-old male golden retriever was diagnosed with leukopenia caused by canine parvovirus in February 2019. Clinical examination showed the body temperature rose to 39.2 degrees, the pulse rate was 110 beats/minute and the breathing was 30 beats/minute. Further blood test showed a significant decrease in white blood cells and neutrophils. It was finally diagnosed as CPV passing the CPV test strip. The case was treated with routine antiviral therapy and fluid supplementation while canine AD-MSCs were intravenously transplanted. The stem cells (1×106 cells per kilogram of body weight) were diluted in normal saline containing 1% canine serum albumin (Blood biotech, China) and transplanted into the sick dog once a day for 3 consecutive times. Biochemical indicators at 2, 3 days after treatment were tested to determine the treatment effect. Control group was another male golden retriever identified with parvovirus infection in the same litter. Clinical examination showed the body temperature rose to 38.5 degrees, the pulse rate was 110 beats/minute and the breathing was 22 beats/minute. The control case was treated with routine antiviral therapy and fluid supplementation.