Name: isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs
Uploaded: 2016-07-02T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.

Animal experiments were authorized by the Animal Center for Biomedical Experimentation at Gyeongsang National University.
1. Preparations for the Animal Procedure
<ol>
	<li>Prepare the adult female minipigs for the non-invasive collection of MSCs from bone marrow and synovial fluid. Perform the clinical examination of the minipigs one day prior to anesthesia and sample collection.
	<ol>
		<li>Provide clinically normal animals for physical examinations, which include body temperature, respiratory rate, and heart rate, and for the observation of the pigs&#39; behavior and hematological statuses, using complete blood count tests. 
		Note: Parameter intervals for clinically healthy minipigs are 11 - 29/min for the respiration rate, 68 - 98/min for the heart rate, and 37 - 38 &#176;C for the body temperature.</li>
	</ol>
	</li>
	<li>Remove all edible bedding from the cage during the preoperative fasting period of 6 -&#160;12 hr prior to the administration of anesthesia for young and adult minipigs, 3 hr of fasting should be observed for the neonates. Provide drinking water until the time of anesthesia.</li>
</ol>
2. Preparation of the Animals for Anesthesia
<ol>
	<li>Medicate the minipigs before the administration of anesthesia with acepromazine (0.1 mg/kg), medetomidine (0.03 mg/kg), and atropine (0.04 mg/kg).
	<ol>
		<li>Inject all medications intramuscularly (IM) using a 21 G needle with a length of at least 30 mm for adult minipigs. Perform the IM injection 1-3 cm behind the ear into the lateral cervical muscle or into the thigh muscle. 
		Note: Perform all the procedures without eliciting fear or stress in the animals or requiring rough handling to safely inject the premedication.</li>
	</ol>
	</li>
	<li>Place the minipigs in a dorsal-recumbent&#160;position.</li>
	<li>Fifteen minutes after the administration of the premedication, initiate anesthesia using 1.5% isoflurane administered through inhalation.
	<ol>
		<li>Continue the inhalation of isoflurane (1.5%) through an endotracheal tube, followed by an increase in concentration to 2.5%, with oxygen (1.5 L/min) as the carrier gas.</li>
	</ol>
	</li>
	<li>Continuously monitor the heart rate, body temperature, and percutaneous blood oxygen saturation (SpO2) of the minipigs during anesthesia. Use a circulating water blanket at a temperature of 38 - 39 &#176;C to maintain body temperature. Apply an ophthalmologic ointment to protect the eyes from drying out during anesthesia.</li>
	<li>Follow Guedel&#39;s classification to assess the depth of anesthesia13.</li>
</ol>
3. Preparations for Cell Isolation and Cultivation
<ol>
	<li>To cultivate the MSCs derived from bone marrow and synovial fluid, prepare 500 ml of advanced Dulbecco&#39;s modified eagle medium (ADMEM) supplemented with 10% fetal bovine serum (FBS), 1% Glutamax, 10 ng/ml of basic fibroblast growth factor (bFGF), and 1.0% penicillin-streptomycin (10,000 IU and 10,000 &#181;g/ml, respectively, Pen-Strep).
	<ol>
		<li>Incubate the ADMEM at 38.5 &#176;C in a humidified atmosphere of 5% CO2 within a CO2 incubator.</li>
	</ol>
	</li>
	<li>Prepare 500 ml of Dulbecco&#39;s phosphate buffer saline (DPBS) according to the manufacturer&#39;s instructions for cell isolation and washing.</li>
</ol>
4. Collecting Bone Marrow from the Minipigs
<ol>
	<li>Select an area approximately 15 x 15 cm in size on the iliac crest (Figure 1), and remove the hair from this area using sterilized safety razors or electric clippers. Once the minipig is positioned, alternately scrub the procedure site three times with the following sterilization agents: povidone iodine and 70% ethanol. After the area is thoroughly dry, apply sterile drapes.</li>
	<li>Pre-coat the 10 ml syringe with 100 U/ml of heparin by rinsing it with 3 - 4 ml of heparin before ejecting the anti-coagulating agent. 
	Note: Perform this step to avoid coagulation in the syringe during bone marrow aspiration.</li>
	<li>Extract a minimum of 5 ml of bone marrow from the iliac crest bone of the minipig under anesthesia, using a bone marrow extractor (3.0&#160;&#215; 100 mm) tightly attached to the heparin pre-coated 10 ml syringe.</li>
</ol>
5. Isolation of Cells Extracted from the Bone Marrow and In Vitro Cultivation
<ol>
	<li>Isolate the cells from the extracted bone marrow.
	<ol>
		<li>Dilute the extracted bone marrow with an equal volume of DPBS in a 15 ml conical tube at room temperature.</li>
		<li>Add 3 ml of density centrifugation media (Ficoll-Paque) to the 15 ml conical tube.</li>
		<li>Dilute a 4 ml layer of the bone marrow with DPBS, which is placed on the density centrifugation media without mixing, and centrifuge it at 400 &#215; g for 40 min&#160;at room temperature.</li>
		<li>Harvest the layer of mononuclear cells (MNCs) from the buffy coat between the two layers containing the plasma (top layer) and the density centrifugation media (bottom layer), and transfer the MNCs into a 15 ml conical tube.</li>
		<li>Dilute the transferred MNCs with 7 - 8 ml of DPBS and centrifuge them at 500 &#215; g for 10 min&#160;at room temperature. Discard the supernatant and wash the cells twice with DPBS.</li>
		<li>Suspend the cell pellet in 2 ml of ADMEM and transfer it to a 35 mm tissue culture dish. Incubate the culture dish at 38.5 &#176;C in a humidified atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>In vitro cultivation of cells isolated from bone marrow.
	<ol>
		<li>After 48 hr, aspirate the medium with the cells that have not attached to the dish, and replace 2 ml of the ADMEM and incubate it at 38.5 &#176;C in a humidified atmosphere of 5% CO2. Change the medium every third days.</li>
		<li>At 70 - 80% confluency, wash the cells with DPBS, dissociate them with 1 ml of 0.25% trypsin/EDTA, and incubate them at 37 &#176;C for 3 - 5 min until the cells detach.</li>
		<li>Harvest the cells using 10 ml of ADMEM, transfer them into a 15 ml conical tube, and centrifuge them at 300 &#215; g for 5 min at room temperature. Discard the supernatant.</li>
		<li>Suspend the harvested cells in 1 ml of fresh ADMEM and count them using a hemocytometer.</li>
		<li>Place the cells in a 5 - 7.5 x 105/25T flask containing 4 ml of ADMEM and incubate them at 38.5 &#176;C in a humidified atmosphere of 5% CO2. Change the medium every third day.</li>
	</ol>
	</li>
</ol>
6. Collection of Synovial Fluid from the Minipigs
<ol>
	<li>Similar to the procedure for bone marrow aspiration, select an appropriate area of approximately 15 x 10 cm (length/width) over the femoral joint, and remove the hair using sterilized safety razors or electric clippers, follow by sanitization procedures (step 4.1).</li>
	<li>Insert a 3 ml syringe with a 23 G needle into the joint after the palpation landmarks (patella and tibia crest).
	<ol>
		<li>Apply gentle suction to the syringes for the aspiration of synovial fluid in the joint. The approximate volume of synovial fluid collected from a joint is 0.5 - 1 ml, but this varies according to the size of the minipig or the joint. Flush through DPBS in the synovial cavity if the collected volume of synovial fluid is too small.</li>
		<li>Immediately withdraw the syringe to avoid contamination with blood.</li>
	</ol>
	</li>
</ol>
7. Cell Isolation from the Collected Synovial Fluid and In Vitro Cultivation
<ol>
	<li>Isolate the cells from the aspirated synovial fluid.
	<ol>
		<li>Dilute the aspirated synovial fluid with an equal volume of DPBS in a 15 ml conical tube at room temperature.</li>
		<li>To remove debris, filter the diluted synovial fluid using a 40 &#181;m nylon cell strainer.</li>
		<li>Add 10 ml of DPBS to the tube containing the filtered synovial fluid and centrifuge it at 400 &#215; g for 10 min at room temperature. Discard the supernatant and wash the cells twice with DPBS.</li>
		<li>Suspend the cell pellet in 2 ml of ADMEM, place the cells on a 2 - 3 x 105&#160;cells/35 mm tissue culture dish with 2 ml of ADMEM, and incubate them at 38.5 &#176;C in a humidified atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Repeat step 5.2 for the in vitro cultivation of isolated synovial fluid-derived MSCs.</li>
</ol>
8. Post-procedure Care of the Minipigs
<ol>
	<li>If needed, close the skin at the bone marrow extractor injection site with a single suture using a skin stapler, after the bone marrow collection.</li>
	<li>After the procedure is completed, administer an intramuscular injection of enrofloxacin (5 mg/kg) and meloxicam (0.2 mg/kg) to the minipigs once a day for 3 days.</li>
	<li>After the procedure is completed, disinfect the bone marrow and synovial fluid collection site with 0.1% povidone iodine once a day for 3 days. Carefully observe daily to monitor for signs of complications (e.g., swelling, pus, or redness).</li>
</ol>
9. Cell Phenotyping Using Flow Cytometry
<ol>
	<li>When the MSCs are 70 - 80% confluent at passages 3 - 5, wash the cells with DPBS and treat them with 1 ml of 0.25% (v/v) Trypsin/EDTA. Then, incubate them at 37 &#176;C for 3 - 5 min until the cells are detached.</li>
	<li>Harvest the cells using 10 ml of DPBS, transfer them into a 15 ml conical tube, and centrifuge them at 300 &#215; g for 5 min at room temperature. Discard the supernatant solution.</li>
	<li>To fix the cells, suspend the cell pellet in 10 ml of a 3.7% formaldehyde solution and keep them at 4 &#176;C for one day before the analysis.
	<ol>
		<li>One day after fixing, wash the cells with DPBS twice using centrifugation at 300 &#215; g for 5 min each time. Discard the supernatant solution.</li>
		<li>Suspend the cell pellet in 4 ml of DPBS, which is supplemented with 1% (w/v) bovine serum albumin (BSA), and incubate the pellet for 1 hr at 4 &#176;C to block non-specific Fc-mediated interactions.</li>
		<li>Distribute 500 &#181;l aliquots of the cell suspension into 1.5 ml tubes.</li>
		<li>For non-conjugated antibodies (CD29 and Vimentin), wash the cells by centrifugation at 300 &#215; g for 3 min. Discard the supernatant solution.</li>
		<li>Suspend the cell pellet in 500 &#181;l of a 1:100 dilution of the primary antibody and incubate it for 1 hr at 4 &#176;C.</li>
		<li>Wash the cells twice with 1% bovine serum albumin by centrifugation at 300 &#215; g for 5 min each time. Discard the supernatant solution.</li>
		<li>Suspend the cell pellet in 500 &#181;l of a 1:100 dilution of the fluorescein isothiocyanate (FITC)-conjugated secondary antibody and incubate it for 1 hr in the dark at 4 &#176;C.</li>
	</ol>
	</li>
	<li>For the FITC-conjugated antibodies (Isotype, CD34, CD44 and CD45), wash the cells by centrifugation at 300 &#215; g for 3 min. Discard the supernatant solution.
	<ol>
		<li>Suspend the cell pellet in 500 &#181;l of the 1:100 FITC-conjugated antibody and incubate it for 1 hr in the dark at 4 &#176;C.</li>
		<li>For both staining conditions, wash the cells twice with DPBS by centrifugation at 300 &#215; g for 5 min each time. Discard the supernatant solution.</li>
		<li>Suspend the cells in 500 &#181;l of DPBS, transfer them into a 5 ml round-bottom tube, and analyze the results using a flow cytometer8.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no conflict of interest.

Minipigs were used to establish MSCs isolation from bone marrow and synovial fluid. To eliminate various physiological conditions, such as age, gender, and disease, minipigs from isogenic background donors were chosen to accurately evaluate the cell source dependent characterization. Pigs are known to be anatomically, physiologically, and genetically similar to humans, and in particular, minipigs can produce size-matched organs; therefore, they can be used as a substitute donor species for xenotransplantation14,15. Regarding the recently suggested immunomodulatory property of MSCs, indoleamine 2, 3-dioxygenase (IDO) and nitric oxide synthase (NOS) were used with MSCs to mediate immunosuppression, which is species-specific, although MSCs from pigs and humans have the same IDO9. Thus, a pig model as a MSCs donor might provide important insights of the immune responses of MSCs for human therapies. Furthermore, the research of the porcine stem cells plays an important role in the biological understanding of in vitro processes and their various preclinical applications in humans16,17. Bone marrow and synovial fluid can be non-invasively harvested from minipigs. The entire procedure, from anesthesia to sample collection, reduced the stress level of systemic or local organs; therefore, minipigs can be used as recipients for autologous transplantation. In this experiment, MSCs were isolated from the bone marrow and synovial fluid of minipigs, based on the confirmation of synovial-fluid-derived MSCs with immunosuppression capacities derived from an RA mouse model in a previous study8
Collection of Bone Marrow from the Minipigs
In pigs, adult progenitor cells derived from bone marrow are multipotent and have induced neovascularization and cardiomyocyte regeneration. The age of a donor is associated with increased fat content and decreased proliferation and differentiation of the bone marrow; therefore, bone marrow is not the best source of MSCs in aged animals. The iliac crest in minipigs is the preferred site for bone-marrow-derived MSC isolation because it is broad and is a low-risk procedure site for sciatic nerve damage. In the case of aged or overweight minipigs, fat-enriched thick muscles may be present on the iliac crest. Under such circumstances, a stabbing incision on the skin is necessary and the bone marrow biopsy needle must be inserted properly, which can be difficult to apply at the iliac crest. Failure to find the correct biopsy site may lead to failed bone marrow aspirations because the needle becomes blocked by the bone or may not be able to reach the bone marrow inside the iliac crest. Therefore, the biopsy needle must be checked properly, and knowledge of the thickness of soft tissue and the cortex of the bone of the iliac crest is required before beginning sample collections.
Aspiration of Synovial Fluid from the Joints of Minipigs
Synovial fluid exists in the cavity of synovial joints, and the volume of synovial fluid is usually increased in inflamed joints. Synovial-fluid-derived MSCs are taken from preferred candidate sites for cartilage regeneration, both in vivo and in vitro. Additionally, they have been shown to elicit immunomodulatory properties in RA8. Synovial fluid is not only an easily accessible source but also has valuable mesenchymal properties. Limitations of using synovial fluid as a source for MSCs are the different volumes found in the joints based on the varying sizes and pathological conditions of the joints and the difficulty in using a 3 ml syringe for synovial fluid aspiration from the joints. Therefore, the size of the syringe should be 5 - 10 ml with high pressure. In addition, flushing of the synovial fluid with 1 - 2 ml of DPBS from a syringe is preferable. By using this method, a large number of cells can be obtained, even from small spaces in the joints.
Characterizations of the Bone-marrow- and Synovial-fluid-derived MSCs of Minipigs
In humans, the morphologies of synovial-fluid- and synovial-membrane-derived MSCs from osteoarthritis patients are similar but narrower than the morphology of bone-marrow-derived MSCs12. However, in in vitro cultures, the morphologies of both synovial-fluid-derived MSCs and bone-marrow-derived MSCs were observed to be similar in this experiment. In the researchers&#39; previous study, the proliferation ability of synovial-fluid-derived MSCs was high, compared to that of bone-marrow-derived MSCs8.
MSCs are established from various tissue sources in adult donors; therefore, non-invasively obtained tissue-source-derived MSCs are valuable for autologous stem cell therapy. The cell source is dependent on the characterizations and capacities of the disease or purpose of the stem cell therapy. In pigs, adult progenitor cells derived from bone marrow are multipotent and have induced neovascularization and cardiomyocyte regeneration18,19. MSCs have been mainly isolated from bone marrow, but also from the periosteum, skeletal muscles, synovium, umbilical cords, and dental tissues, although obtaining cells from these tissues is not easy and can require additional surgical procedures for the patients. However, the volume of synovial fluid usually increases in the joints of osteoarthritis or RA patients, and biopsies of these increased synovial fluids are used to diagnose and treat patients. The number of MSCs also increases in the synovial fluid of patients with degenerated cartilage and osteoarthritis20.
In this study, the cell phenotypes of bone-marrow- and synovial-fluid-derived MSCs were evaluated, and both MSCs presented similar levels of expression for cell surface markers. Therefore, cells isolated from synovial fluid and confirmed as MSCs have similar phenotypes to bone-marrow-derived MSCs in minipigs. The researchers&#39; previous research evaluated other cell surface markers, such as CD90 and the major histocompatible and complex class II (MHC II), and confirmed a capacity for multi-lineage differentiation into the osteocytes, adipocytes, chondrocytes, and neurons of synovial-fluid-derived MSCs from minipigs8. In humans, the cell surface antigen profile of synovial-fluid-derived MSCs is similar to that of synovial-membrane-2,21 and bone-marrow-derived MSCs. However, CD34 and CD45 expressions were not detected in MSCs derived from synovial fluid in temporomandibular joint disorder patients. Thus, the microenvironment in the joint may affect the cell phenotype of MSCs derived from synovial fluid. In this experiment, the cells were obtained from the bone marrow and synovial fluid of minipigs using non-invasive methods. Both types of in vitro cultured cells were confirmed as MSCs, and they had similar cell phenotypes and shared compatible mesenchymal characteristics. However, synovial-fluid-derived MSCs showed a greater potential for autologous stem cell therapy for not only cartilage and bone regeneration but also for immunosuppression of osteoarthritis and RA.

Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.

<table><tbody><tr><td>Advanced Dulbecco&amp;rsquo;s modified Eagle medium (ADMEM)</td><td>Gibco</td><td>12491-023</td><td>MSC culture medium</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190-144</td><td>Cell washing medium, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>Component of MSCs medium</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>Component of MSCs medium</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>10378-016</td><td>Component of MSCs medium</td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Simga</td><td>F0291</td><td>Component of MSCs medium</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A6003</td><td>Component of MSCs medium</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td>Cell dissociation reagent&amp;nbsp;</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>Component of MSCs medium</td></tr><tr><td>Isotype antibody</td><td>BD Pharmingen</td><td>BD 550616</td><td>Isotype Control</td></tr><tr><td>CD29 antibody</td><td>BD Pharmingen</td><td>BD 552369</td><td>Integrin beta-1 MSCs marker</td></tr><tr><td>CD44 antibody</td><td>BD Pharmingen</td><td>BD 553133</td><td>Cell surface glycoprotein MSCs marker</td></tr><tr><td>CD45 antibody</td><td>BD Pharmingen</td><td>BD 340664</td><td>Hematopoietic stem cells marker</td></tr><tr><td>CD34 antibody</td><td>BD Pharmingen</td><td>BD 555821</td><td>Hematopoietic stem cells marker</td></tr><tr><td>CD90 antibody</td><td>BD Pharmingen</td><td>BD 555595</td><td>Thy-1 membrane glycoprotein MSCs marker</td></tr><tr><td>MHC Class II antibody</td><td>Santa Cruz</td><td>SC-32247</td><td>Antigen presenting cells marker</td></tr><tr><td>Vimentin antibody</td><td>Sigma</td><td>Sigma-S6389</td><td>Type III intermediate filament MSCs marker</td></tr><tr><td>Alkaline phosphatase</td><td>Promega</td><td>S3841</td><td>Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td></tr><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-1440-02</td><td>Density gradient centrifugation</td></tr><tr><td>Cell strainer</td><td>BD Falcon</td><td>352340</td><td>40 &amp;micro;m nylon cell strainer</td></tr><tr><td>15-ml polystyrene conical tube</td><td>BD Falcon</td><td>351095</td><td>Sample collection and cell isolation</td></tr><tr><td>35 mm dishes</td><td>Nunc</td><td>153066</td><td>Cell culture dish</td></tr><tr><td>Bone marrow extractor</td><td>GmbH Medizintechnologie, Germany</td><td>1145-1W010</td><td>TrokaBone, 3.0 x 100 mm</td></tr><tr><td>Hematocritchamber</td><td>Marinfeld</td><td>640130</td><td>Cell counting chamber</td></tr><tr><td>Atropine</td><td>Jeil Pharmaceutical Co, South Korea</td><td></td><td>Atropine sulfate Hydrate</td></tr><tr><td>Acepromazine</td><td>Samu Median, South Korea</td><td></td><td>Pre-anaesthetic sedative and antiemetic drug</td></tr><tr><td>Medetomidine</td><td>Pfizer</td><td></td><td>Anesthetic and analgesic drug</td></tr><tr><td>Enrofloxacin</td><td>Bayer Healthcare, Germany</td><td></td><td>Anti-biotics</td></tr><tr><td>Meloxicam</td><td>Over Veterinary Medicine, Argentina</td><td></td><td>Anti-inflammatory and analgesic drug</td></tr><tr><td>Isoflurane</td><td>Hana pharm, South Korea</td><td></td><td>Inhalational anesthesia</td></tr><tr><td>Povidone iodine&amp;nbsp;</td><td>Korea Pharma, South Korea</td><td></td><td>Sterilization agent</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023</td><td>Sterilization agent</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td>Anti-coagulating agent</td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td>Cell fixation agent</td></tr><tr><td>Ophthalmologic ointment&amp;nbsp;</td><td>Pfizer</td><td></td><td>Oxytetracycline HCl with polymyxin B sulfate</td></tr><tr><td>Circulating water blanket</td><td>Gaymar Industries</td><td></td><td>Warming system during and after anesthesia</td></tr><tr><td>Skin stapler</td><td>Covidien, USA</td><td></td><td>Suture skin closure</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Forma</td><td>3111</td><td>Cell culture Equipment</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td>Cell analyzer</td></tr><tr><td>Minipig</td><td>PWG Genetics Korea, Ltd.</td><td>T-type</td><td>Miniature pig</td></tr></tbody></table>

isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

Establishment of MSCs Derived from the Bone Marrow and Synovial Fluid of Minipigs
MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro (Figure 2). Syringe aspiration of synovial fluid is simple, and it is possible to obtain sufficient adherent cells during in vitro cultivation of the primary cells. The morphology of synovial-fluid-derived MSCs is similar to that of bone-marrow-derived MSCs, and both MSCs have a fibroblastic spindle shape and are homogeneously adherent. They were observed to have a positive expression for alkaline phosphatase (AP) staining, after the third subculture was complete.
Evaluation of the Cell Phenotype of MSCs Derived from the Bone Marrow and Synovial Fluid of Minipigs
Assays of the cell surface markers were performed to create a phenotype of the bone-marrow and synovial-fluid-derived MSCs of minipigs. Both types of MSCs expressed significant amounts of positive cell surface markers, such as CD29, CD44, and Vimentin (&#8805;96 - 99% of the positive cells), whereas the expressions of CD34 and CD45 were negative and low (&#8804;2% of the positive cells) (Figure 3). These results were identical to those obtained from distinctive MSCs, including cells isolated from synovial fluid, and there were no significant differences in the cell surface markers among these MSCs. The cells isolated from bone marrow and synovial fluid have been confirmed as MSCs in our previous study8. Both bone marrow- and synovial fluid-derived MSCs expressed stem cell transcriptional factors (Oct3/4, Nanog, and Sox2); moreover, an in vitro differentiation ability has been confirmed not only into the mesenchymal lineage (osteocytes, adipocytes, and chondrocytes), but also neural cells by cytochemical staining and detections of cell-specific gene expression8.
<img alt="Figure 1" src="/files/ftp_upload/54077/54077fig1.jpg" /> 
Figure 1. Location of the iliac crest of the minipig. The red arrow represents the site on the iliac crest used for the bone marrow collection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54077/54077fig2.jpg" /> 
Figure 2. MSCs derived from the bone marrow and synovial fluid of minipigs. (A) The homogeneous morphology of primary cultures of MSCs revealed a fibroblastic shape. (B) Staining with mixture of 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (BCIP/NBT) revealed AP activity in the MSCs at passage 3. Scale bar: 100 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54077/54077fig3.jpg" /> 
Figure 3. Analysis of the cell surface markers of MSCs derived from the bone marrow and synovial fluid of minipigs at passage 3. The cell surface markers of MSCs were identified using a flow cytometry analysis, and MSCs were identified as being positive for CD29, CD44, and Vimentin and negative for CD34 and CD45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

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Nanyang Technological University

Research

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Medicine

10.3K Views.  Gyeongsang National University. A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Video: Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Developmental Biology

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Bioengineering

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Biology

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

Unveiling Inflammatory Arthritis&#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

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