Name: isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs
Uploaded: 2016-07-02T14:00:00
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, respira...

<ol>
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W., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perspectives+for+porcine-to-human+xenografts.">The perspectives for porcine-to-human xenografts.</a> Comp Immunol Micro biol Infect Dis. 32 (2), 91-105 (2009).</li><li>Zeng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+adult+progenitor+cells+from+swine+bone+marrow.">Multipotent adult progenitor cells from swine bone marrow.</a> Stem Cells. 24 (11), 2355-2366 (2006).</li><li>Zeng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Sekiya, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+in+synovial+fluid+increase+in+the+knee+with+degenerated+cartilage+and+osteoarthritis.">Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.</a> J Orthop Res. 30 (6), 943-949 (2012).</li><li>Mochizuki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+chondrogenic+potential+of+fibrous+synovium-+and+adipose+synovium-derived+cells+compared+with+subcutaneous+fat-derived+cells:+Distinguishing+properties+of+mesenchymal+stem+cells+in+humans.">Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: Distinguishing properties of mesenchymal stem cells in humans.</a> Arthritis Rheum. 54 (3), 843-853 (2006).</li></ol>

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</...

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 border="0" cellpadding="0" cellspacing="0" width="499">
	<tbody>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">Advanced Dulbecco&#8217;s modified Eagle medium (ADMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">12491-023</td>
			<td style="width:143px;">MSC culture medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">14190-144</td>
			<td style="width:143px;">Cell washing medium, free of Ca2+/Mg2+,</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:136px;">Fetal bovine serum (FBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">16000-044</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">35050-061</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Penicillin/streptomycin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">10378-016</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:143px;">Simga</td>
			<td style="width:79px;">F0291</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Bovine serum albumin (BSA)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">A6003</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Trypsin-EDTA</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">25200-072</td>
			<td style="width:143px;">Cell dissociation reagent&#160;</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">&#946;-mercaptoethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">M7522</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Isotype antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 550616</td>
			<td style="width:143px;">Isotype Control</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:136px;">CD29 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 552369</td>
			<td style="width:143px;">Integrin beta-1 MSCs marker</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">CD44 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 553133</td>
			<td style="width:143px;">Cell surface glycoprotein MSCs marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD45 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 340664</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD34 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555821</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">CD90 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555595</td>
			<td style="width:143px;">Thy-1 membrane glycoprotein MSCs marker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">MHC Class II antibody</td>
			<td style="width:143px;">Santa Cruz</td>
			<td style="width:79px;">SC-32247</td>
			<td style="width:143px;">Antigen presenting cells marker</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Vimentin antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">Sigma-S6389</td>
			<td style="width:143px;">Type III intermediate filament MSCs marker</td>
		</tr>
		<tr height="115">
			<td height="115" style="height:115px;width:136px;">Alkaline phosphatase</td>
			<td style="width:143px;">Promega</td>
			<td style="width:79px;">S3841</td>
			<td style="width:143px;">Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Ficoll-Paque</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:79px;">17-1440-02</td>
			<td style="width:143px;">Density gradient centrifugation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Cell strainer</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">352340</td>
			<td style="width:143px;">40&#181;m nylon cell strainer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">15-mL polystyrene conical tube</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">351095</td>
			<td style="width:143px;">Sample collection and cell isolation</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:136px;">35 mm dishes</td>
			<td style="width:143px;">Nunc</td>
			<td style="width:79px;">153066</td>
			<td style="width:143px;">Cell culture dish</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:136px;">Bone marrow extractor</td>
			<td style="width:143px;">GmbH Medizintechnologie, Germany</td>
			<td style="width:79px;">1145-1W010</td>
			<td style="width:143px;">TrokaBone, 3.0x100mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Hematocritchamber</td>
			<td style="width:143px;">Marinfeld</td>
			<td style="width:79px;">640130</td>
			<td style="width:143px;">Cell counting chamber</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:136px;">Atropine</td>
			<td style="width:143px;">Jeil Pharmaceutical Co, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Atropine sulfate Hydrate</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:136px;">Acepromazine</td>
			<td style="width:143px;">Samu Median, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Pre-anaesthetic sedative and antiemetic drug</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Medetomidine</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anesthetic and analgesic drug</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:136px;">Enrofloxacin</td>
			<td style="width:143px;">Bayer Healthcare, Germany</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-biotics</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Meloxicam</td>
			<td style="width:143px;">Over Veterinary Medicine, Argentina</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-inflammatory and analgesic drug</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Isoflurane</td>
			<td style="width:143px;">Hana pharm, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Inhalational anesthesia</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:136px;">Povidone iodine&#160;</td>
			<td style="width:143px;">Korea Pharma, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Ethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">E7023</td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Heparin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">H3393</td>
			<td style="width:143px;">Anti-coagulating agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Formaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">F8775</td>
			<td style="width:143px;">Cell fixation agent</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:136px;">Ophthalmologic ointment&#160;</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Oxytetracycline HCL with polymyxin B sulfate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Circulating water blanket</td>
			<td style="width:143px;">Gaymar Industries</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Warming system during and after anesthesia</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:136px;">Skin stapler</td>
			<td style="width:143px;">Covidien, USA</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Suture skin closure</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">CO2 Incubator</td>
			<td style="width:143px;">Thermo Forma</td>
			<td style="width:79px;">3111</td>
			<td style="width:143px;">Cell culture Equipment</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:136px;">Flow cytometer</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Cell analyzer</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:136px;">Minipig</td>
			<td style="width:143px;">PWG Genetics Korea, Ltd.</td>
			<td style="width:79px;">T-type</td>
			<td style="width:143px;">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 morpholo...

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, ...

The overall goal of this procedure is the non-invasive collection of synovial fluid and bone marrow from minipigs to collect mesenchymal stem cells to be used in regeneration and immunomodulation studies. This method can help answer key questions in the otologist MSC therapy field, such as how to establish the synovial fluid in MSC, and how to characterize them in comparison to bone marrow MSC. The main advantage of this technique is to effectively establish the key properties of synovial fluid and bone marrow-derived MSCs from minipigs, which will be useful for application in patients.The implications of this technique extend toward therapy of various joint diseases, because the population of the synovial fluid-derived MSC in the synovial cavity is significantly increased in athletic patients. Though this method can provide insight into regenerative medicine, it can also be applied to other studies such as autoimmune disease research. Because minipig MSCs are autologous, immunosuppression might interest humans.In addition to the other scientists, demonstrating the procedure will be Hyeon-Jeong Lee and Won-Jae Lee from my level three. To begin, orient an anesthetized minipig to access the iliac crest. Next, depilate a 15-square-centimeter region on the iliac crest, using a razor or clippers.Then, alternately scrub the exposed skin three times with povidone-iodine and 70%ethanol. Once completely dried, apply sterile drapes. Now, pre-coat a ten-milliliter syringe with heparin, using a three-to four-milliliter rinse.This will prevent the bone marrow from coagulating while it is extracted. Then, precisely at the iliac bone crest, tightly attach a bone marrow extractor to the heparin-coated syringe, and extract at least five milliliters of bone marrow. To isolate the cells from the extracted bone marrow, first dilute it with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.Then, load three milliliters of density centrifugation media into a second 15-milliliter conical tube. Next, carefully transfer four milliliters of the diluted bone marrow onto the density centrifugation media to create two-solution layers. Now, centrifuge the tube at 400 Gs for 40 minutes at room temperature.The desired MNCs are collected in the central white layer. Aspirate just this layer. Eject the MNC layer into a 15-milliliter conical tube, and dilute it with seven to eight milliliters of DPBS.Then, centrifuge the cells at 500 Gs for ten minutes at room temperature. Discard the supernatant, and wash the palleted cells twice with ten milliliters of DPBS, repeating the previous centrifugation cycle. After the two washes, we suspend the MNCs in two milliliters of ADMEM.Then, load the cells onto a 35-millimeter tissue culture dish, and incubate them at 38.5 degrees Celsius in a humidified 5%carbon dioxide incubator. After 48 hours, aspirate the medium along with the unattached MNCs. Then, add back two milliliters of fresh ADMEM, and continue the incubation, changing the medium every three days.When the cells reach 70 to 80%confluency, wash the culture with fresh DPBS. Then, dissociate the cells, using one milliliter of 0.25%Trypsin-EDTA for three to five minutes at 37 degrees Celsius. Collect the cells in ten milliliters of ADMEM, and transfer them into a 15-milliliter conical tube.Then, spin the cells down at 300 Gs for five minutes at room temperature, and discard the supernatant. Now, suspend the harvested MNCs in one milliliter of fresh ADMEM, and measure their density. Finish with plating between 1/2 and three-quarter million cells into T25 flasks with four milliliters of ADMEM.Incubate the flasks just as the plates, and change the medium every third day. Prepare a 15x10 centimeter section of skin over the femoral joint, as was done when preparing for bone marrow collections. Then, palpate for the patella and tibia crests below the skin to identify the synovial joint, and insert a three-milliliter syringe with a 23-gauge needle, precisely into the joint.Using gentle suction, extract between 1/2 and one milliliter of fluid, and immediately withdraw the needle. If the synovial fluid volume is too small, then flush the cavity with DPBS to collect synovial fluid with DPBS. The volume of DPBS to use varies with the size of the synovial cavity.The optimal volume of synovial fluid is limited in healthy joint. The volume varies with the size and pathological condition of its joint. To isolate the cells from the synovial fluid, dilute the fluid with an equal volume of DPBS in a 15-milliliter conical tube at room temperature.Next, we move the debris by filtering the dilution through a 40-micron nylon cell strainer. Then, add ten more milliliters of DPBS, and centrifuge the suspension at 400 Gs for ten minutes at room temperature. Next, discard the supernatant, and wash the cells two times, using DPBS.We suspend the washed cell pallet in two milliliters of ADMEM. Then, proceed with culturing the cells as was demonstrated with the MSCs. MSCs were successfully isolated from the bone marrow and articular synovial joints of the minipigs and expanded in vitro.The morphology of synovial fluid-derived MSCs is similar to that of bone marrow-derived MSCs:both have a fibroblastic spindle shape and are homogeneously adherent. The MSCs showed positive expression for alkaline-phosphatase staining after the third subculture was completed. Next, surface markers were analyzed in both cultures.As expected, both types of MSCs significantly expressed CD29, CD44, and Vimentin. By contrast, expression of CD34 and CD45 were negative and low. These results were identical to those obtained through distinctive MSCs, including cells isolated from synovial fluid.After watching this video, you should have a good understanding of how to non-invasively aspirate synovial fluid and bone marrow in order to isolate their MSC from minipigs and how to perform the comparison and phenotyping of this MSC. While attempting this procedure, it's important to remember to locate the objective point on the iliac crest and the synovial, determined by careful palpation. Following this procedure, other methods like in models of osteoarthritis or rheumatoid arthritis can be performed in order the answer additional questions like whether not only from marrow MSC but also synovial fluid MSCs have a same kind capacity for cartilage or bone initiation and immunosuppressions.After its development, this technique advanced autologous therapy of joint diseases by helping to characterize MSCs derived from attacked or inflamed joint in arthritis patients.

isolation-cellular-phenotyping-mesenchymal-stem-cells-derived-from

Research

JoVE Journal

Medicine

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Synovial Fluid Analysis to Identify Osteoarthritis

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white blood cell (WBC) count, help define the non-inflammatory nature of SF, which is a hallmark of OA. In patients with OA, WBC in SF samples usually does not exceed 2000 cells per microliter, and the percentage of inflammatory cells, such as neutrophils, is very low or absent. Calcium crystals are frequent in SF collected from OA patients. Although their role in the pathogenesis of OA remains unclear, they have been associated with a mild inflammatory process and a more severe disease progression. Recently, calcium crystals have been described in both the early and late stages of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment. The overall goal of SF analysis in OA is two-fold: to ascertain the non-inflammatory degree of SF and to highlight the presence of calcium crystals.

Synovial fluid analysis under transmitted, polarized, and compensated light microscopy is used to evaluate the inflammatory or non-inflammatory nature of a sample through simple steps. It is particularly useful in osteoarthritis to detect calcium crystals and identify a more severe subset of osteoarthritis.

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...

Dissection and Isolation of WJ-MSCs from the Human Umbilical Cord

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton's Jelly for Enhanced MSC Production

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...