PROCEDEU DE IZOLARE, CULTIVARE ȘI IDENTIFICARE EFICIENTĂ A CELULELOR STEM MEZENCHIMALE DIN MĂDUVA OSOASĂ ÎN CONDIȚII DE LABORATOR [310082]

[anonimizat] A [anonimizat], [anonimizat], MD, PhD, [anonimizat], [anonimizat], PhD, [anonimizat], the Republic of Moldova

*Corresponding author: [anonimizat]

Manuscript received….; revised manuscript ……

Abstract

Background: Bone marrow mesenchymal stem cells (MSC) have a wide application in domain of regenerative medicine. MSC also represents a particular interest for in vivo and in vitro laboratory tests. Of great importance is utilisation of a suitable bone marrow extraction technique that can provide a sufficient number of MSC to perform laboratory tests fara a aduce grave daune sanatatii animalului de laborator without seriously affecting the health of the laboratory animal. [anonimizat].

Materials and Methods: The study was conducted in rabbits (n = 9), [anonimizat] 3.39 ± 1.27 ml of bone marrow. The nucleated bone marrow cells were separated through centrifugation using concentration gradient. With culture medium specific for stem cells the MSC were multiplied during 2 passages. From th 2-nd passage in each case ware taken by 1×106 MSC and subjected to the differentiation by chondrocytes line for 20 days. [anonimizat] O and Toluidine blue with Fast Green

Results: There was a strong correlation between the volume of collected bone marrow and the number of days required to achieve a 70-80% MSC confluency (p=0,01), also the MSC isolated from bone marrow extracted from a [anonimizat] O and Toluidine blue with Fast Green (p˂0,001).

Conclusions: The volume of bone marrow harvested from rabbit iliac bone is sufficient to obtain a large number of MSC for the laboratory tests in vitro and in vivo with autocells and as a standard method for MSC identification can be used just terminal differentiation in the specialized cell.

Key words: [anonimizat], rabbit, [anonimizat], autocells.

Introduction

Mesenchymal stem cells are multipotent cells that can differentiate into different cell lines depending on the micromedium in which they are stored [1, 3, 14, 15, 16]. These cells had a [anonimizat], tendon, meniscus, degenerative lesions of the locomotor apparatus nervous system and internal organs [5, 6, 7, 8, 9, 10, 11, 13]. Methods of bone marrow harvesting from laboratory animals for isolation and cultivation of MSC for in vivo and in vitro tests is diverse. Most common method of bone marrow extractions is slaughtering of the animals with further jet flushing of diaphyses and epiphyses of long tubular bones [18, 19]. However, this method allows the use of stem cells obtained for in vitro tests and as allogeneic cells for in vivo tests. Another way is the aspiration of bone marrow from the metaphysical areas of the long tubular bones after perforating the bone with a drill bit [2, 4, 17]. It requires a deep anesthesia and a lot of time for the procedure. This method allows experiments on the animals with autocells, but the deep anesthesia, the surgery that certainly will not be the last and the postoperative recovery period may endanger the experiments success due to suffering or even death of the animal [20]. Another way is performing of a short-term superficial anesthesia with local potency and harvesting the bone marrow from the iliac bone [1, 3], similar procedure performed in humans [21].

Materials and methods

The study was performed on 9 house rabbits 4 to 5 months old, 6 females and 3 males, with average weight of 3.78 ± 0.25 kg, in which, from one iliac bone ware taken 3,39 ± 1,27 ml of bone marrow, then with a concentration gradient HiSep LSM 1077 (HiMedia, India) the nucleated cells were separated. Then, the cells were cultured with mesenchymal stem cell expansion medium HiMesoXL (HiMedia, India) in incubator at 37°C, 5%CO2 (SMART CELL, Heal Force) during the first 2 passages. In order to demonstrate the presence of mesenchymal stem cells in cultured cells, the MSC from the 2-nd passage were differentiated into chondrocytes using the chondrocyte differentiation medium HiChondroXL (HiMedia, India). The differentiated chondrocyte aggregates were histologically examined by cartilage specific staining. The research on rabbits received a positive decision at the ethics committee meeting from 14.12.2016 with No. 31.

Cell media preparation

Culture medium for mesenchymal stem cells (MSC) was obtained by adding 11.4 ml of component B in 500 ml of component A of the medium for the mesenchymal stem cells expansion HiMesoXL (HiMedia, India) and 5 ml of antimycotic antibiotic solution (HiMedia, India) [24].

The medium for chondrocyte differentiation is obtained by adding component B to 100 ml of component A of the chondrocyte differentiation medium HiChondroXL (HiMedia, India) and 1 ml of antimycotic antibiotic solution (HiMedia, India) [23].

The media was prepared according to the manufacturer's instructions, with further sterilisation by filtration with 0.22μm pores diameter PES filters (Sofra, China) and stored in the refrigerator at 4-8°C.

Isolation and cultivation of MSC from bone marrow harvested from the iliac bone.

On the day of bone marrow harvesting, the not feeded animals were weighed, followed by intramuscular injection at the hip region of 5 mg/kg xylazine and 2 mg/kg diazepam solutions. With a trimmer, the fur was removed from the dorsal part of the basin followed by aseptic processing with betadine and 70% alcohol solution. To potentiate the anesthesia, at the level of the iliac wing were injected 4 ml of 1% lidocaine. The operator field was delineated with sterile bedding, after which was prepared a 5 ml syringe with 1250-2000 U heparin. With an 18G needle accompanied by a trocar, making slow, rotating movements, parallel to the iliac bone plane, from anterior to posterior, the first cortical of the iliac bone was perforated. The trocar was extracted, after which the bone marrow was aspirated with the heparinized syringe [1, 3] (fig. 1). The syringe with bone marrow went to the laboratory for processing and the animal was taken back to the vivarium.

Fig. 1. Bone marrow harvesting from a rabbit iliac bone. Preparing the animal for bone marrow harvest (a, b), sterile work space delimitation (c), instruments preparation for the procedure (d), perforation of iliac bone cortex with 18G needle (e) and bone marrow obtaining (f, g).

In the laboratory, the syringe with bone marrow was introduced into the laminar flow hood. The concentration gradient HiSep LSM 1077 (HiMedia, India) and PBS (Lonza, Belgium) preventively, were heated in the water bath. In a 15 ml sterile tube the concentration gradient was poured in a equal volume with harvested bone marrow. The bone marrow was shifted in a 10 or 20 ml syringe containing the same volume of PBS (Lonza, Belgium). After homogenization, the PBS with bone marrow have been poured cautiously on the concentration gradient from the 15 ml tube without mixing. The tube was centrifuged at 400 x g for 15 minutes followed by removement of upper 2/3 of platelets and adipocytes layer, the mononuclear cell layer was collected in a separate tube along with 1/3 of the remaining overlying layer and the upper 1/3 of concentration gradient layer [22]. Then to the tube with mononuclear cells was added PBS (Lonza, Belgium) till the tube was filled, followed by a careful pipetting and centrifuged at 170 x g for 10 minutes (fig. 2). The supernatant was removed and the centrifugation has been repeated after pipetting the cells with 10 ml of culture medium. After centrifugation the cells were resuspended in 5 ml culture medium, placed in a 25 cm2 cell culture flask (Nunc, Denmark) without counting the mononuclear cells. The cell culture flask was introduced into the incubator (SMART CELL, Heal Force) at 37°C with 5% CO2 with the change every 2-3 days of a half of the nutrition medium (fig. 3).

Fig. 2. Processing of harvested bone marrow. The 15 ml tube with bone marrow mixed with PBS located upon the concentration gradient in a ratio of 1: 1: 1 (a), separation on layers after centrifugation (b) and the bone marrow nucleated cell layer separated in another tube in the washing process (c).

Fig. 3. Isolation of MSC from bone marrow x60. Concentrate of bone marrow nucleated cells (a), apparition of MSC fusiform cells attached to the flask cell culture surface after 2 days of cells culture (b) and the 70-80% confluence of the MSC after 5 days of culture (c).

After a 70-80% confluence of the cells attached to the bottom of cell culture flask, the culture medium was poured out the cells were washed twice with PBS (Lonza, Belgium) after which 2 ml of 0.25% trypsin-EDTA solution was added into the flask, followed by addition of 2 ml trypsin-EDTA 0.25% solution into the flask. The flask was placed in the incubator for 3 to 5 minutes, after which 2-3 coups were applied to the flask, followed by microscopic examination of detached cells. Tripsinization was stopped with 3 ml of soybean trypsin inhibitor (Lonza, Belgium). The cells suspension was centrifuged at 170 x g for 5 minutes. After supernatant decantation, 5 ml of culture medium was added and the cells gently pippeted. The cells was counted cells with a haemocytometer and viability was assessed with 0,4% trypan blue (Sigma, UK) exclusion. Then all cells were placed in 75 cm2 cell culture flasks (Nunc, Denmark) at a density of 1×104 ± 1×103 cells/cm2, with culture medium complete changement every 2 days, until a 80-90% confluence (fig. 4). After trypsinization and cells counting with trypan blue exclusion (Sigma, UK), from each culture have been isolated 1×106 cells and differentiated on chondrocytes line and the other cells were frozen by 5×105 cells/ml of FBS (Lonza, Belgium) with 10% DMSO (OriGen Biomedical, Germany) for in vitro laboratory tests.

Fig. 4. MSC culture in 2nd passage x60. MSC attached to the culture surface with traces of bone marrow cells after 24 hours of culture (a), CSM on the 3rd day of culture (b) and the 90% confluence of MSC at 5th day of culture (c).

Chondrocyte differentiation from MSC

The MSC differentiation potential is considered to be a functionally reliable criterion for their identification and their distinction from preadipocytes, preosteocytes or prechondrocytes [1, 3, 24]. As a cells differentiation medium was used the medium for chondrocytes line differentiation HiChondroXL (HiMedia, India).

In a 15 ml polypropylene tube ware introduced 1×106 cells/ml of MSC culture medium. The tube was centrifuged at 45 x g for 10 minutes, followed by supernatant elimination and addition of 1 ml of chondrocyte differentiation medium HiChondroXL (HiMedia, India). The cells were gently resuspended in medium and the tube was centrifuged once again. The tube was placed in a stand and was introduced into the incubator (SMART CELL, Heal Force) with a gently opened lid at 37°C, 5% CO2 and wet environment without disturbing the cell pellet. The chondrocyte differentiation medium was changed every 48 hours for 20 days [24]. At 5th-7th days of differentiation, at the tube bottom was observed formation of spherical or oval shape aggregates (fig. 5). After 20 days the aggregates were introduced into 10% buffered formaldehyde and stained with Hematoxylin-Eosin and specific staining for cartilage with Safranin O and Toluidine Blue with cu Fast Green [25].

Fig. 5. MSC differentiation on chondrocyte line in 15 ml polypropylene tubes. The aspect of 1×106 MSC before the differentiation on chondrocyte line (a) and formation of chondrocytes aggregates (b).

The data statistical analysis was carried out using Excel and SPSS 17.0 programs.

Results

The period of time needed for bone marrow harvest from the beginning of anesthesia is 36±3 minutes. During the bone marrow harvesting and after that, complications in experimental animals were not recorded. Once the cells from 1st passage reached to a 70-80% confluence they were trypsinized, thus, the average duration of the first passage cultivation was 7±1 days with a strong correlation between the volume of harvested bone marrow and the number of days required to achieve a 70-80% cells confluency (p=0,01). In Table 1 are presented the results of the MSC obtention from rabbit bone marrow resulted from cultivation in the first 2 passages.

Table 1

In all cases 2nd passage was cultivated for 5 days sufficient time to achieve a 80-90% cellular confluence in all cases. So the average duration of cells cultivation during the first 2 passages was 12±1 days, with a surprisingly 100% cell viability in all cases.

In the process of chondrocyte differentiation, the formation of cell aggregates was determined which were of irregular spherical shape attached to the bottom of the tube, which later detached and floated freely through the medium. Also in the first days of differentiation, the cells could be easy dispersed by pipetting, but after they became floated aggregates the cells were no longer dispersed at pipetting. Even if cells aggregates consisted of the same number of cells, they could have different sizes, which varies between 1.5 – 3 mm in diameter (fig. 5).

At the histological examination with Hematoxylin-Eosin a rich cellularity of obtained aggregates was determined, highlighted by a high density of cells nuclei and extracellular matrix formation (fig. 6).

Fig. 6. Hematoxylin-Eosin staining of chondrocytes aggregates x80.

At Toluidine blue staining with Fast Green, the obtained structure was intensely colored in purple and blue. Because the cells are arranged in conglomerates, their nuclei can not be distinguished due to the overlapping of a large number of them, at the same time the blue color represents the cartilage extracellular matrix synthesized by chondrocytes (fig. 7).

Fig. 7. Staining with Toluidine blue and Fast Green of formed aggregates x80.

After staining with Safranin O, a large number of darkened nuclei were determined and the extracellular matrix secreted by the cells was steined in red, this being specific to cartilaginous tissue (fig. 8).

Fig. 8. Colorarea cu Safranin O a agregatelor formate x80.

The identification of obtained cells aggregates was positive at the specific staining for cartilaginous tissue in all cases (n=9) and is statistically significant (p˂0,001).

Discussions

Bone marrow harvesting from rabbit iliac bone serves as an effective way to isolate and cultivate mesenchymal stem cells. This method allows performing in vivo tests with the rabbits own MSC, without subjecting the animal to great suffering which could adversely affect the results of the experiments [20]. Numerous cases of MSC isolation from long tubular bones after rabbit sacrifice are described in the literature [5, 7, 18, 19], or aspiration of bone marrow from metaphyseal areas of long tubular bones, like femur or even tibia tibie [2, 4, 7, 17].

The iliac bone is smaller than the femoral bone respectively, the volume of harvested bone marrow will be smaller. However, the thickness of the iliac bone in the cortical perforation site, in an adult rabbit, ranges between 4.3-4.8 mm (fig. 9), respectively the iliac bone can be easily penetrated with a 18G needle and the volume of harvested bone marrow may reach to 4.5-5 ml, just from one side, without harming the animal's health and expose it to risks which can cost us time and money [20]. At the same time, it must be taken into account that the obtained volume of bone marrow is more than enough to obtain in a relatively short period of time, 12±1 days, a number of 4116667±464354,4 cells which is quite important.

Fig. 9. The comparative dimensions of the iliac and femoral bones. Rabbit femoral and iliac bones (a). Assessment of iliac bone (b) and femoral bone (c) thickness.

According to literature data it is known that in the bone marrow the number of mesenchymal stem cells is very small, between 0,01 – 0,001% of total number of bone marrow nucleated cells [1], or 1/10000-1/100000 according to other sources [26, 27, 28]. MSC can be multiplied for 500 times during 50 generations, finally getting billions of cells [26, 27], also, from the 6th passage of in vitro culture, MSC lose their stem cells characteristics and the ability to differentiate [27, 28]. In other words, if we continue cultivating the obtained cells further in passages at a density of 8×103 cells/cm2, at the 5th passage we would have well over 1 billion cells with differentiation potential. Therefore even 1 ml of bone marrow taken from a single iliac bone in rabbits, represents a sufficient volume to get a large number of MSC capable to differentiation for in vitro or in vivo tests on rabbits, which is groundless denied and ignored in the literature [2, 4, 7, 8, 15, 16, 18].

Stem cells identifying is an important step in working with them. At the moment, the most common ways to identify MSC are RT-PCR, cell differentiation by adipogenic, osteogenic and chondrogenic pathway [1, 3], flow cytometry [1, 26], immunofluorescence microscopy [26, 28]. In our research we identified bone marrow MSC only through chondrogenic differentiation pathway. Therefore, according to the criteria of the International Society of Cellular Therapy, MSC have not been fully identified, but in our opinion this is sufficient, because bone marrow MSC are multipotential cells with terminal differentiation potential in specialized cells. However, the differentiation on the chondrocytes line was confirmed by specific staining for cartilaginous tissue with Safranin O and Toluidine blue with Fast Green [25, 29].

Disclaimer

The authors of this article did not benefit and will not benefit from the goods from commercial agents or manufacturers for using the media purchased from them.

Bibliographie

Ninu A.R., Swapan K.M., Shiva Kumar M.U., Sandeep K., Sangeetha P., Deepka K., Saurabh G., Abhisek S., Naveen K. Isolation, proliferation, characterization and in vivo osteogenic potential of bone-marrow derived mesenchymal stem cells (rBMSC) in rabbit model. In: Indian Journal of Experimental Biology. 2017; 55(2): 79-87.

Phedy, Dilogo I.H., Jusuf A.A., Kholinne E., Efendi Z. Iliac crest and femoral bone marrow as the source of plastic-adherent cells. In: Medical Journal of Indonesia. 2011; 20:100-4.

Gugjoo M.B., Amarpal, Kinjavdekar P., Prasad A.H., Ansari M.M., Pawde MotiRam A., Taru G.S. Isolation, Culture and Characterization of New Zealand White Rabbit Mesenchymal Stem Cells Derived from Bone Marrow. In: Asian Journal of Animal and Veterinary Advances. 2015; 10(10): 537-548.

Piñero EçaI L., Belmonte Ramalho R., Sousa O., Gomes P.O., Pontes P., Teixeira Ferreira A., Vaccari Mazzetti M.P. Comparative study of technique to obtain stem cells from bone marrow collection between the iliac crest and the femoral epiphysis in rabbits. In: Acta Cirúrgica Brasileira. 2009; 24(5): 400-404

Kalamegam G., Memic A., Budd E., Abbas M., Mobasheri A. A comprehensive review of stem cells for cartilage regeneration in osteoarthritis. In: Advances in Experimental Medicine and Biology. 2018; 1089:23-36. doi: 10.1007/5584_2018_205.

Zhao L., Kaye A.D., Abd-Elsayed A. Stem Cells for the Treatment of Knee Osteoarthritis: A Comprehensive Review. In: Pain Physician. 2018; 21:229-241 ISSN 1533-3159

Walmsley G.G., Ransom C.R, Zielins R.E., Leavitt T., Flacco S. J., Hu S.M., Lee S.A., Longaker T.M., Wan C.D. Stem Cells in Bone Regeneration. In: Stem Cell Reviews. 2016; 12(5): 524–529. doi: 10.1007/s12015-016-9665-5

Nauth A., Schemitsch H. E. Stem cells for the repair and regeneration of bone. In: Indian Journal of Orthopedics. 2012; 46(1): 19–21. doi: 10.4103/0019-5413.91630

Po Yee Lui P. Stem cell technology for tendon regeneration: current status, challenges, and future research directions. In: Stem Cells Cloning. 2015; 8: 163–174. doi: 10.2147/SCCAA.S60832

Ahmad Z., Wardale J., Brooks R., Henson F., Noorani A., Rushton N. Exploring the application of stem cells in tendon repair and regeneration. In: Arthroscopy. 2012; 28(7): 1018-29. doi: 10.1016/j.arthro.2011.12.009

McLauchlan D., Robertson N.P. Stem cells in the treatment of central nervous system disease. In: Journal of Neurology. 2018; 265(4): 984–986. doi: 10.1007/s00415-018-8818-7

Abdelwahid E., Kalvelyte A., Stulpinas Aurimas., Teixeira de Carvalho K. A., Guarita-Souza L.C., Foldes G. Stem cell death and survival in heart regeneration and repair. In: Apoptosis. 2016; 21(3): 252–268. doi: 10.1007/s10495-015-1203-4

Cantz T., Manns P. M., Ott M. Stem cells in liver regeneration and therapy. In: Cell Tissue Research. 2008; 331(1): 271–282. doi: 10.1007/s00441-007-0483-6

Augello A., De Bari C. The Regulation of Differentiation in Mesenchymal Stem Cells. In: Human Gene Therapy. 2010; 21(10):1226-38. doi:10.1089/hum.2010.173.

Panadero J.A., Lanceros-Mendez S., Ribelles J.L. Differentiation of mesenchymal stem cells for cartilage tissue engineering: Individual and synergetic effects of three-dimensional environment and mechanical loading. In: Acta Biomaterialia. 2016; 33:1-12. doi: 10.1016/j.actbio.2016.01.037.

Solchaga L.A., Penick K.J., Welter J.F. Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells: Tips and Tricks. In: Methods in Molecular Biology. 2011; 698: 253–278. doi: 10.1007/978-1-60761-999-4_20

Weidong Z., Fangbiao Z., Hongcan S., Rongbang T., Shi H., Gang Y., Shu P., Fei S., Xingchen L. Comparisons of Rabbit Bone Marrow Mesenchymal Stem Cell Isolation and Culture Methods In Vitro. In: PLoS One. 2014; 9(2): e88794. doi: 10.1371/journal.pone.0088794

Mohammed R.H., Jasim A. M., Almanseekanaa L.H. Isolation and characterization of bone marrow mesenchymal stem cells from rat and rabbit: A modified method. In: Kerbala Journal of Pharmaceutical Sciences. 2012; 3

Hao L., Li-Kun W., Xiao-Fei J., Jie H., Hui Z., Shi-Zhan Z., Guang-Hua Y. Isolation, culture and induced differentiation of rabbit mesenchymal stem cells into osteoblasts. In: Experimental And Therapeutic Medicine. 2018; 15: 3715-3724. doi: 10.3892/etm.2018.5894

Naderi M.M., Sarvari A., Milanifar A., Boroujeni S.B., Akhondi M.M. Regulations and Ethical Considerations in Animal Experiments: International Laws and Islamic Perspectives. In: Avicenna Journal of Medical Biotechnology. 2012; 4(3): 114–120.

Patterson T.E., Boehm C., Nakamoto C., Rozic R., Walker E., Piuzzi S.N., Muschler F.G. In: Journal of Bone and Joint Surgery Am. 2017; 99(19): 1673–1682. doi: 10.2106/JBJS.17.00094

http://himedialabs.com/TD/LS001.pdf

http://himedialabs.com/TD/AL512.pdf

http://himedialabs.com/TD/AL523.pdf

Schmitz N., Laverty S., Kraus V.B., Aigner T. Basic methods in histopathology of joint tissues. In: Osteoarthritis and Cartilage. 2010;18: S113-S116. doi: 10.1016/j.joca.2010.05.026

Jones E.A., Kinsey S.E., English A., Jones R.A., Straszynski L., Meredith D.M., Markham A.F., Jack A., Emery P., McGonagle D. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. In: Arthritis & Rheumatology. 2002; 46(12): 3349-60. doi: 10.1002/art.10696

Nasef A., Fouillard L., El-Taguri A., Lopez M. Human bone marrow-derived mesenchymal stem cells. In: Libyan Journal of Medicine. 2007; 2(4): 190–201. doi: 10.4176/070705

Bonab M.M., Alimoghaddam K., Talebian F., Ghaffari S.H., Ghavamzadeh A., Nikbin B. Aging of mesenchymal stem cell in vitro. In: BMC Cell Biology. 2006; 7: 14. doi: 10.1186/1471-2121-7-14

Hoemann C., Kandel R., Roberts S., Saris D. B.F., Creemers L., Mainil-Varlet P., Méthot S., Hollander A.P., Buschmann M.D. International Cartilage Repair Society Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials. In: Cartilage 2011; 2(2): 153–172. doi: 10.1177/1947603510397535