Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

doi:10.21203/rs.3.rs-64097/v1

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Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

https://doi.org/10.21203/rs.3.rs-64097/v1

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published 01 Mar, 2021

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BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications.

RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. No adverse events were noted in two cases treated with intravenously AD-MSCs.

CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

Large Animal Medicine

Small Animal Medicine

canine

cell therapy

mesenchymal stem cells

adipose

banking

Mesenchymal stem cells (MSCs) which were first discovered by Friedenstein from bone marrow derived cells are a type of self-renewing cells harboring the potential to differentiate into diverse tissues, such as bone, cartilage, muscle, fat and tendon[1–5]. At present, in human clinical medicine, a variety of clinical trials with human MSCs are ongoing for the treatment of immunological diseases and degenerative diseases[6–9]. Currently, it has been reported that MSCs could be isolated from various tissues other than bone marrow[10], such as adipose[11], umbilical cord[12], dental pulp[13], placenta[14] and amniotic membrane[15]. Adipose-derived Mesenchymal stem cells (AD-MSCs) have gained in popularity due to the ease and abundance of harvesting adipose tissue, greater capacity of self-renewal multipotency and paracrine immunomodulatory[11, 16–18].

As one of the most common companion animals, some breeds of canine including beagles are also experimental animals. With the improvement of living standards, diseases of aging in canine have become more common clinically, especially chronic diseases, such as osteoarthritis, hip dysplasia, diabetes, pancreatitis and tendonopathy[19, 20]. Meanwhile, there are no particularly effective treatments for these diseases, which not only affect the quality of life of the affected animals, but also require the owners to invest a lot of energy, time and money. Currently, many related reports have confirmed that AD-MSCs therapy in a variety of canine diseases in clinical trials have shown initial results[21–25].

As one kind of superior cytotherapy, canine AD-MSCs therapy has great clinical application potential in various chronic diseases treatment[26, 27]. Although the technology of canine AD-MSCs isolation and expansion at laboratory scale is relatively mature, manufacturing a therapeutic cell-based product is and will continue to be challenging[28, 29]. In this paper, we established a standardized operating procedure for manufacturing canine AD-MSCs, which encompasses tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. The establishment of canine AD-MSCs bank was expected to provide an adequate source of seed cells for the cell therapy of canine diseases and the research of tissue engineering products.

Cell Isolation,Culture and Cryopreservation

Approximately 2–6 × 10⁶ nucleated cells were obtained from adipose tissue (n = 12, range 2–5 grams) after collagenase type I digestion and seeded into 1–2 100 mm cell -culture dishes. After 48 h of growth, the vast majority of AD-MSCs were attached to the culture plate and exhibited fibroblast-like morphology (Fig. 1). The cells grew to 80% − 90% confluence after 5–7 days and were passaged for the first time. After second passages with a 1:3 split ratio, 0.64–2.67 × 10⁷ AD-MSCs passage 2 were harvested and cryopreserved at the end of first expansion period. Each batch of AD-MSCs must passed the in-process test and be given a unique identifier before cryopreservation. Viability of the freshly harvested cells was greater than 90% in all cases. Basic data for the MSC expansions are shown in Table 3.

Table 3

The number of harvested cells in different passages
No.	Size(g)	Harvested Cells of P0 (10⁶)	Harvested Cells of P1 (10⁶)	Harvested Cells of P2 (10⁶)
CAD-001	3.2	3.7	10.5	26.7
CAD-002	4.1	4.2	12.3	24.3
CAD-003	2.1	2.2	4.1	7.8
CAD-004	2.4	4.3	8.3	17.3
CAD-005	3.2	3.2	4.7	9.3
CAD-006	3.2	4.1	9.2	10.4
CAD-007	4.5	4.7	12.2	26.7
CAD-008	1.2	4.3	8.9	19.2
CAD-009	1.9	3.0	6.4	12.6
CAD-010	2.4	4.1	9.4	19.9
CAD-011	6.1	5.8	10.7	23.5
CAD-012	3.1	5.9	12.7	25.9

Characterization and Quality Controls

AD-MSCs presented subsequently fibrous or fusiform shape and showed fibroblast-like adherent growth (Fig. 1).

The Cells of passage 2 and passage 5 were highly-expressed mesenchymal stem cell surface markers CD29, CD44 and CD90, while for the lowly-expressed haematopoietic stem cells surface markers CD34 and leukocyte common antigen CD45. The positive expression rate for CD29, CD44 and CD90, in was > 95%, and the positive expression rate for CD34 and CD55 was < 2% (Table 4 and Fig. 2).

Table 4

Surface marker expression of AD-MSCs
Surface marker	Expression (%)
Surface marker	Passage 2	Passage 5
CD 29	99.35 ± 0.48	99.25 ± 0.59
CD 34	0.18 ± 0.06	0.29 ± 0.31
CD 44	99.22 ± 0.59	98.27 ± 0.73
CD 45	0.47 ± 0.38	0.54 ± 0.28
CD 90	98.32 ± 0.99	98.08 ± 1.43

The ability of adipogenic and osteogenic differentiation in vitro was detected respectively by Oil red O and alizarin red staining. The adipogenic differentiated AD-MSCs were visualized by staining with Oil red-O on day 15. Calcium nodules were observed and stained red by alizarin red in osteogenic induction groups on day 17(Fig. 3).

The population doubling time and Colony-Forming unit-fibroblasts capacity of AD-MSCs was used to assess proliferative ability. The population doubling time was 22.03 ± 2.30 hours in passage 2 and 23.62 ± 1.67 hours in passage 5. The Colony-Forming unit-fibroblasts capacity of AD-MSCs of in passage 2 and passage 5 were 15.20 ± 2.77% and 16.20 ± 3.70%, respectively (Fig. 4).

Sterility, mycoplasma, and endotoxin test were performed before cryopreservation and release of final product, as well as by random testing of culture supernatants in the course of the expansion. All the pre-cryopreservation cells and final products were free of microbial and mycoplasma contamination. Endotoxin levels of pre-cryopreservation cells and final products were lower than 0.5 EU/ml.

Cell Therapy and Case Report

Case 1

One week after AD-MSCs transplantation treatment, the sick dog successively showed objective improvement in mental state and eating normally. The blood creatinine and blood phosphorus levels were measured to fall within the normal range, and the urea content was significantly decreased and close to the normal range (Table 5). AD-MSCs transplantation can effectively treated leptospira-induced acute renal failure.

Table 5

The blood biochemical before and after AD-MSCs transplantation treatment
	Prior Treatment	2 d after Treatment	3 d after Treatment	5 d after Treatment	7 d after Ttreatment	Reference Range
UREA (mmol/L)	108.4	90.5	66.4	34.2	18.4	2.5–9.6
CREA (µmol/L)	463	303	176	137	106	44–169
PHOS (mmol/L)	8.91	6.22	2.66	1.6	1.43	0.81–2.19
UREA, carbamide; CREA, creatinine; PHOS, blood phosphorus

Case 2

The case showed an unexpected response to stem cell therapy. After intravenous transplantation of AD-MSCs, the spirit state of the infected dog improved on the day of treatment, and the number of white blood cells increased on the second day and returned to normal on the third day (Table 6). There were no adverse reactions in response to AD-MSCs therapy.

Table 6

Hemogram levels before and after AD-MSCs transplantation treatment
	Prior Treatment	2 d after Treatment	3 d after Treatment	Reference Range
WBC (10⁹/L)	0.9	1.1	7.3	6.0–17.0
Lym (10⁹/L)	0.2	0.4	2.5	5.1–10.8
Mon (10⁹/L)	0	0	0.4	0-1.8
Gran (10⁹/L)	0.7	0.7	4.4	4.0-12.6
WBC, white blood cell; Lym, lymphocyte; Mon, monocyte; Gran, granulocyte

AD-MSCs are a type of adult stem cell deriving from adipose tissues with self-renewal ability and multidirectional differentiation potential[30]. As an attractive source of MSCs, adipose tissue usually contains far more MSCs than other sources contain[31]. AD-MSCs have become the focus of considerable interest in regenerative tissue engineering due to their easy access and availability in large quantities. The different methods for isolation and culture of canine AD-MSCs has also been established across different countries and different laboratories[22, 24, 26]. Currently, MSCs manufacture including isolation, culture and identification methods are labor intensive, with many time-consuming steps[3, 32]. A complex set of processes introduces the risk of microbial infection and changes in biologic properties[33]. Accordingly, all steps of cell manufacture for clinical application must be performed based on good manufacturing practice to achieve a reasonable safety and quality[34]. The establishment of canine AD-MSCs bank operating in accordance with good manufacturing practice standards will accelerates the clinical application of canine stem cell therapy and the development of stem cell industrialization in pet market.

In this study, we tried to establish a standardized operating process encompasses tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion for clinical grade canine AD-MSCs manufacturing. Adipose tissue samples were aseptically collected from abdomen fat of healthy donor canine under general anaesthesia. Donor canine peripheral blood was then taken for the examination of infectious diseases including RV, CDV, ICHV, CPV, CCV, fungi and bacteria. Subsequent process can be carried out after the qualification is confirmed, which will serve as the initial basis for the study of cell quality. Using mechanical disruption combined with collagenase type I digestion, AD-MSCs could be successfully isolated and further seeded centrifugally into cell culture dishes in our optimum culture conditions.

After thawing and expansion, AD-MSCs showed all properties characteristic for mesenchymal stem cells outlined by the International Society for Cellular Therapy (ISCT) for defining MSCs[35]. The cells were in a fibrocyte-like form and had a spindle-shape, and were arranged in a whirlpool pattern. The cells formed a monolayer of homogenous bipolar spindle-like cells with a whirl-pool-like array. The immunophenotype analysis displayed that the cells positively expressed of CD29, CD44, and CD90 and the lack of CD34, and CD45. In addition, they demonstrated the ability of osteogenic differentiation and adipogenic differentiation.

The manufacturing of AD-MSCs as cell therapy products for veterinary clinical application should be performed with corresponding controls to ensure its safety and quality[36, 37]. The AD-MSCs have undergone safety testing including sterility, and mycoplasma and results found to meet specifications. There were no bacterial, fungal, or mycoplasma contaminations observed in any of our 12 cultures. With a small number of cells and a limited number of injections, the administration of AD-MSCs was demonstrated intravenously without any immediate adverse events.

In comparison with stem cells derived from other sources, AD-MSCs have the advantages of convenient material acquisition, fewer ethical issues, low damage and abundant sources[38, 39]. Our previous study indicated that canine AD-MSCs had the highest proliferation activity vis-à-vis four other sources-derived MSCs in vitro. On average, the number of AD-MSCs doubled every day in our cell culture system [40]. The higher rates of cell proliferation help to harvest more numbers of MSCs in the same unit of time, thus saving cell production costs and times. In addition, the establishment of AD-MSCs banks may accelerate the veterinary clinical application of stem cell therapy and the development of pet stem cell businesses.

In summary, we successfully established a standardized process for manufacturing and banking canine AD-MSCs for clinical use and confirmed adipose tissues representing an appealing source of MSCs for cell therapy. It is hoped that the clinical experimental research and application of canine AD-MSCs in regenerative medicine will benefit the majority of sick animals and contribute to the health of pet dogs.

Adipose Tissue Collection

Adipose tissue of the canine abdomen was collected during routine clinically indicated surgery at Affiliated Animal Hospital of Foshan University. 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

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 and osteogenic differentiation were examined using MSCs Adipogenic Differentiation Kit (Cyanogen, China) and MSCs Osteogenic Differentiation Kit (Cyanogen, China) following the manufacturer’s protocol for each kit. Cells were stained with Oil Red O solution to assess adipogenic differentiation and alizarin red solution to assess osteogenic 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.

Cell Therapy

Case 1 A 14-year-old male teddy dog was diagnosed with acute renal failure caused by Leptospira in March 2018. 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. After 3 days of conventional treatment, the dog received stem cell therapy for 2.7 × 10⁶ AD-MSCs (1 × 10⁶ cells per kilogram of body weight) intravenously.

Case 2 A 2-month-old male golden retriever was diagnosed with leukopenia caused by canine parvovirus in February 2019. 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.

Statistical analysis

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.

AD-MSCs adipose-derived Mesenchymal stem cells

CCV canine coronaviruses

CDV canine distemper virus

CFU-F Colony-Forming unit-fibroblasts

CPV canine parvovirus

DMEM Dulbecco's Modified Eagle's Medium

FBS fetal bovine serum

FITC fluorescein isothiocyanate

ICHV infectious canine hepatitis virus

Me²SO dimethyl sulfoxide

MSCs mesenchymal stem cells

PE phycoerythrin

RV rabies virus

Td population doubling time

Ethics approval and consent to participate

All procedures in the present study were approved by the Animal Ethics Committee of Foshan University and written informed consent was obtained from all donors.

Consent for publication

Written informed consent for publication was obtained from all participants.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by Natural Science Foundation of Guangdong Province (grant no. 2017A030313171).

Authors’ contributions

Bingyun Wang and Shengfeng Chen designed the study. Huina Luo and Dongsheng Li performed the experiments and analysed the data. Zhisheng Chen collected the samples. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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published 01 Mar, 2021

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Editorial decision: Major revision
26 Sep, 2020
Review #1 received at journal
25 Sep, 2020
Review #2 received at journal
21 Sep, 2020
Reviewer #2 agreed at journal
17 Sep, 2020
Reviewer #1 agreed at journal
05 Sep, 2020
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Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

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Journal Publication

Version 1

Abstract

Figures

Introduction

Result

Cell Isolation,Culture and Cryopreservation

Characterization and Quality Controls

Cell Therapy and Case Report

Discussion

Conclusion

Materials And Methods

Adipose Tissue Collection

Cell Isolation and Culture

Cryopreservation

Thawing and Expansion

Characterization and Quality Control

Cell Therapy

Statistical analysis

List Of Abbreviations

Declarations

References

Status:

Journal Publication

Version 1