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. 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.
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).
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.
Case 1 A 14-year-old male Teddy dog weighing 2.7 kg was diagnosed was diagnosed with acute renal failure caused by Leptospira in March 2018. Clinical symptoms showed an increase in the number of white blood cells and neutrophils, and the red blood cells and hematocrit were decrease. Urea nitrogen, creatinine, blood phosphorus and alkaline phosphatase are elevated. In addition, according to the body inflammation had been strong, anemia, dehydration and other symptoms, the dog kidney function was judged to be impaired. The case showed little response to sodium lactate ringer or sodium bicarbonate for regulating electrolyte balance and cephalosporin, enoxacin or doxycycline for anti-inflammatory sterilization. Cell therapy were performed for the symptoms did not improve after 3 days of conventional treatment. The canine AD-MSCs were diluted in 50 mL of normal saline and then transplanted into the patient by intravenous injection at 1×106 cells per kilogram of body weight. Biochemical indicators were tested at 2, 3, 5, and 7 days after treatment to determine the effect of treatment.
Case 2 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 cases were 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.
SPSS 17.0 software (SPSS, Inc.) was used for statistical analysis. Values are expressed as the means ± standard deviation. Statistical analysis was performed using one-way analysis of variance with the least-significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.