Name: Isolating Stem Cells from Soft Musculoskeletal Tissues (Video) | JoVE Journal
Uploaded: 2010-07-05T17:00:00.000+00:00
Duration: 7 min 49 s
Description: Learn to isolate adult stem cells from skeletal muscle using enzymatic digestion and repeated pre-plating techniques. This method enables characterization via immunofluorescence and flow cytometry for regenerative medicine research.

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The authors are greatly appreciative of Ms. Haiying Pan and Mr. Ian Bellayr for their technical help on cell isolation for the current films and Mr. Kyle Holden and Mr. Jonathan Lucas for their help on slide editing. Many thanks to the following list of persons who have been involved with the development of the modified preplate techniques over the past years: Drs. Burhan Gharaibeh, BaoHong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, Kimimasa Tobita, Ron Jankowski, Makato Ikezawa. We are grateful for the technical assistance from James H. Cummins, Ryan Pruchnic, Joseph Feduska, Rebecca C. Schugar, Haiying Pan and Jessica Tebbets. The author also would like to acknowledge the financial support for this work from the Muscular Dystrophy Association (MDA), the National Institutes of Health (NIH), the Department of Defense (DOD), the Children's Hospital of Pittsburgh of UPMC, the Department of Orthopaedic Surgery, and the University of Pittsburgh.

In general, the complete modified preplate procedure requires 1-2 days of preparation, 1 week of performing the technical procedure, 2-3 days of cell identification with immunocytochemistry, and an additional 2-3 weeks of stem cell population expansion. The equipment required for stem cell culture are similar to that of other cell culture systems which includes a bench-top centrifuge, CO2 incubator, Laminar flow tissue culture hood and an inverted microscope equipped with fluorescence and a digital camera. The isolated stem cells also can be cloned and can be stored long-term in liquid nitrogen for the current or future studies 1,2. All reagents and materials used for this procedure must be sterile and handled aseptically. Step 1: Preparation <ol> <li>Collagen coat tissue culture dishes and flasks: Type I collagen derived from calf skin (0.1 g in 1 liter; Sigma-Aldrich). The plasticware to coat includes: T-25 flasks, 6 or 12 well plates, dishes (60 and 100 mm, BD Falcon) as described before 1,2. Gently shake the plasticware every half hour, for 4 hours to ensure even coating of the collagen. The collagen solution is then removed. And the coated plasticware is then allowed to dry in the tissue culture hood with the UV light and the hood both on, to ensure sterility, and finally recapped or covered for later use.</li> <li>Prepare cell culture medium : 400mL DMEM (DMEM, high glucose; Invitrogen), 50mL Heat Inactivated Fetal Bovine Serum (FBS, Invitrogen), 50mL Horse Serum (HS, Invitrogen), and 5 mL Chick Embryo Extract (CEE; Accurate Chemical Co.) will be vacuum filtered using a disposable 0.22-&mu;m 500-ml sterile filter system (Corning). Sterile penicillin/streptomycin solution (Invitrogen) will be added finally 100 Units/mL. The prepared solutions can be stored at 4&deg;C for at least one month. This solution is used to detach the cells from the flask when splitting cultures or preparing the cells for transplantation.</li> <li>Warm up solutions for cell isolation: The following reagents need to be warmed up to 37&deg;C prior to their use for cell isolation: Hank's Buffered Salt Solution (HBSS, Invitrogen); 0.2% (wt/vol) collagenase-type XI (Sigma-Aldrich), with an average of 3,200 collagen digestion units per mL HBSS; Dispase 2.4 units/1mL (Invitrogen); 0.5% (wt/vol) trypsin (Invitrogen).</li> <li>Sterilize surgical equipment in preparation: Surgical procedure require sterile surgical instruments including: different-sized forceps, micro-scissors, iris scissors and/or scalpel blades. Additionally, 15 and 50-mL polypropylene centrifuge tubes (BD Falcon); Cell strainer (pore size 70 m) (BD Falcon); 18-, 23- and 27-gauge needles (BD PrecisionGlide); and 10- or 20-ml sterile syringes (BD).</li> </ol> Step 2: Tissue biopsies, isolation and mechanical dissociation1-4 The tissue biopsies are performed under a sterile culture hood and include freshly harvested tendons and skeletal muscles are obtained from the tibia or soleus muscles of the hindlimb of wild-type mice (Female C57BL/6J, 4-5 weeks of age or younger, Jackson Laboratory). Any visible connective tissue, blood vessels, and adipose tissue are then removed from the biopsy samples. The tissues of tendon and muscle are then carefully identified under a surgical dissecting microscope to remove remnants of skin and bone and placed in a dish containing cold (4&deg;C) HBSS (supplemented with add 5 % of FBS). The isolated tissues are then separately placed on collagen coated dishes containing cold HBSS and will be minced (&lt;1x1 mm3) into a coarse slurry using micro-scissors and/or scalpel blades. Step 3: Enzymatic digestion of the tissues: The minced tissue is then enzymatically dissociated through a series of steps2,4,5 <ol> <li>Transfer the minced tissue slurries into separate 15-ml tubes and centrifuge at ~3500 rpm at 4 &deg;C for 5 mins.</li> <li>Remove the supernatant, wash with HBSS and repeat the centrifugation again.</li> <li>Remove the supernatant, and digest these slurries by adding 10 ml of prewarmed 0.2% collagenase-type XI (Sigma). Incubate for 60 mins at 37 &deg;C while continuously gently rocking the tubes. Alternatively, shake by hand every 10 min.</li> <li>Centrifuge and resuspend these slurries in 10 ml dispase (2.4 Units/mL, Gibco) solution. Incubate for 45 mins at 37 &deg;C, shaking with hands or gently rocking every 10 min.</li> <li>Centrifuge and re-suspend these slurries in 10 ml of 0.2% trypsin HBSS solution. Incubation will vary based on the extent to which the single cells are released from the tissues which can be performed by observing the suspension under microscope. It is not recommend to exceed 30 mins at 37 &deg;C with inverting the tubes or gently rocking every 10 min.</li> <li>Centrifuge the resulting cells and re-suspend the cell pellet in 10 ml of Proliferation Medium (PM).</li> <li>Dissociate the cell suspension by passing the extract through a series of needles. The cells are then passed 2 times through an 18G, then a 23G, and finally a 27G needle.</li> <li>Pass the cell extract through a 70-&mu;m cell strainer.</li> </ol> Step 4: Preplating to isolate different cell populations based on cell adhesion rate1-3 <ol> <li>Centrifuge and resuspend the cell pellets in Proliferation Medium.</li> <li>Plate the cell mixtures onto a collagen-coated T-25 flask (or 60 mm dish) and mark it as PP1. The cell suspension should be observed as possessing large numbers of refractive nuclei and other debris under the microscope.</li> <li>Incubate at 37 &deg;C in a humidified, 5% CO2 incubator for 2 hours.</li> <li>Transfer nonadherent cells to a new collagen-coated T-25 flask (or 60 mm dish) and mark is as PP2. Add 5 ml of PM into the plate labeled PP1. The early adhering cells which attach to the flask are mostly myofibroblasts3.</li> <li>Return the flasks to the incubator for another 24 hours, transfer nonadherent cells to a new collagen-coated T-25 flask (or dish) and mark it as PP3. Add 5 ml of Proliferation Medium into the plate labeled PP2. These adhering cells which attach to this flask are mostly fibroblasts2,3.</li> <li>Return flasks to the incubator and repeat the procedure in another 24 hours until population PP6 is created. Maintain each group of suspended cells and allow them to proliferate and examining them regularly using an inverted microscope. PP3-PP4 are mostly myoblasts while muscle satellite cells are often seen mostly in PP5 2.</li> <li>Most of the slowly adhering cells typically attach by PP6. At this stage, the cells appear small, round, light refractive and are very sparse in number and are considered to be a stem cell population 1,2.</li> </ol> Step 5: Identification and expansion of isolated stem cells <ol> <li>The isolated PP6 cells are very few in number and need to be maintained in FBS rich Proliferation Medium (20% FBS in DMEM) that is changed daily. Most of the cells present in the PP6 culture die during the following 1-2 weeks of culturing, but a large, healthy PP6 population is usually created after an additional 1-2 weeks of expansion. The stem cells isolated from mice highly express stem cell antigen 1 (Sca1), CD34, fetal liver kinase 1 (Flk1), and other stem markers1,2,6,7 which can be visualized via immunocytochemical staining. These stem cells also exhibit multiple differentiation capacities3,6,8.</li> <li>The isolated cells are maintained and expanded at a low cell density in culture (12-well dishes at 50-100 cells/well or &lt;20-30% confluence in the flask or dish) to avoid differentiation 1,2. Very few cells form clones during expansion, and these cloned stem cells can undergo long time proliferation (LTP) for any other competence studies. The cloned stem cells also can be store in liquid nitrogen using general cell storage procedures e.g. PM medium with 10% DMSO. Make a number of low passage stocks of the stem cells for any other back up studies.</li> </ol>

<ol><li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li>
<li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+novel+population+of+muscle+stem+cells+in+mice%3A+potential+for+muscle+regeneration%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li>
<li>Li, Y. &amp, Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li>
<li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &amp, Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li>
<li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li>
<li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li>
<li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li>
<li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li>
<li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+medicine+based+on+muscle+stem+cells%5BTitle%5D)+AND+%22J+Musculoskelet+Neuronal+Interact%22%5BJournal%5D)">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li>
<li>Gates, C. B., Karthikeyan, T., Fu, F. &amp, Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells%5BTitle%5D)+AND+%22J+Am+Acad+Orthop+Surg%22%5BJournal%5D)">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li>
</ol>

No conflicts of interest declared.

It has been recognized that some adult tissues contain multiple stem cells. Over the past few years of study, stem cells have been detected in the soft musculoskeletal tissues, including skeletal muscle and tendon. This current protocol details a procedure, known as the modified preplate technique, that has been successfully used in our laboratory to isolate adult stem cells from tendons and skeletal muscle. Using the modified preplate technique described above the cells can be divided into several populations based on t...

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>#11995-073</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>#10437-028</td><td></td></tr><tr><td>Heat-inactivated horse serum</td><td>Invitrogen</td><td>#26050-088</td><td></td></tr><tr><td>Chick embryo extract</td><td>Accurate Chemical</td><td>#CE650T-10</td><td></td></tr><tr><td>100U ml penicillin/streptomycin</td><td>Invitrogen</td><td>#15140-122</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Invitrogen</td><td>#14190-250</td><td></td></tr><tr><td>Hank&#039;s Buffered Salt Solution</td><td>Invitrogen</td><td>#24020-117</td><td></td></tr><tr><td>Collagenase type XI 0.2%(wt/vol)</td><td>Sigma-Aldrich</td><td>#C7657</td><td></td></tr><tr><td>Dispase2.4U ml</td><td>Invitrogen</td><td>#17105-041</td><td></td></tr><tr><td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td><td>Invitrogen</td><td>#15400-054</td><td></td></tr><tr><td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td><td>Sigma-Aldrich</td><td>#C-9791</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>#D-2650</td><td></td></tr></tbody></table>

isolating stem cells from soft musculoskeletal tissues

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

The overall goal of this procedure is to isolate adult stem cells from musculoskeletal soft tissues such as skeletal muscle. This is accomplished by first taking a biopsy of the soft tissue, debriding it and chopping it finely. The second step of the procedure is to enzymatically digest the tissue.The third step of the procedure is to isolate the stem cells through repeated pre plating steps. The final step of the procedure is the identification of the isolated stem cells. Ultimately, results can be obtained that show the cells morphological and cellular behaviors using immunofluorescence, microscopy and or flow cytometry.Hi, I'm y Lee from the Lab of Molecular Pathology in the Department of Orthopedic Surgery at the University of Pittsburgh. Today I will show you how to isolate a adult stem cell from the soft tissues of Mus skeletal system such as skeletal muscle. Hello, I'm Dr.Johnny Ward.I'm a professor at the University of Pittsburgh School of Medicine within the Department of Orthopedic Surgery. I've been working, you know, closely with Dr.Young Lee and this procedures to insulate, you know, different type of stem cell from skeletal muscle and alter tissues such as standard. We use the procedures in our laboratory to isolate stem cell for basically research studies on the translational medicine applications.So let's get studied. Let the tissues from which stem cells can be isolated include tendons and skeletal muscles harvested from the tibia or soleus muscles of the hind limb of wild type mice. After removing remnants of skin and bone, place tissues in a dish containing cold HBSS supplemented with 5%FBS.Next, place the tissue separately on collagen coated dishes containing cold HBSS. We'll use skeletal muscle tissues to demonstrate the basic procedures for tissue dissociation and digestion. Use micro scissors and or scalpel blades to mince the skeletal tissues into a coarse slurry pH enzymatic digestion of the tissues.Start by transferring the MIT tissue slurries into separate 15 milliliter tubes in centrifuge at approximately 3, 500 RPM at four degrees Celsius for five minutes. Remove the SANE wash by Resus suspending an HBSS and repeat the centrifugation. After removing the supernat, digest the slurries by adding 10 milliliters of prewarm.0.2%collagenase.Type 11 incubate for 60 minutes at 37 degrees Celsius, shaking the tubes by hand every 10 minutes when the 60 minute incubation is done. Centrifuge and then resuspend the slurries in 10 milliliters of DYS space solution. Incubate for 45 minutes at 37 degrees Celsius, shaking by hand every 10 minutes after the 45 minute incubation centrifuge and resus suspend the slurries in 10 milliliters of 0.2%trips in HBSS solution.We usually incubate for 15 to 20 minutes at 37 degrees Celsius while inverting the tubes every 10 minutes, the incubation is complete. When enough single cells are released from the tissues as illustrated here centrifuge the resulting cells and resus suspend the cell pellet in five milliliters of proliferation. Medium PM dissociate the cell suspension by passing the extract through a series of needles two times through an 18 gauge needle, then once through a 23 gauge needle and finally once through a 27 gauge needle.Lastly, pass the cell extract through a 70 micron cell strainer to begin the prepl technique. Centrifuge the cell extract that was just prepared and resuspend the pellets in proliferation. Medium plate five milliliters of the cell mixture onto a collagen coated T 25 flask and market as PP one incubated 37 degrees Celsius in a humidified 5%carbon dioxide incubator for two hours.After two hours. Transfer the non-adherence cells to a new collagen coated T 25 flask and market as PP two. Add five milliliters of PM into the previous T 25 flask labeled PP one.Return both flasks to the incubator for 24 hours. After 24 hours, transfer non-adherent cells from PP two to a new collagen ENC coated T 25 flask and mark it as PP three. Add five milliliters of proliferation medium into the flask labeled PP two.Return the flasks to the incubator. Repeat the procedure every 24 hours until population PP six. The stem cell population is created.The isolated PP six cells are very few in number and need to be maintained in FBS rich proliferation medium. That has changed daily. Although most of the cells in the PP six culture will die during the following one to two weeks of culturing.A large healthy PP six population is usually created after an additional one to two weeks of expansion. Maintain all other groups of suspended cells as well, allowing them to proliferate and examining them regularly using an inverted microscope. The early adhering cells that attach to the PP one and PP two flasks are mostly fibrotic cells.Cells that adhere to collagen coated flasks after a longer time period within 48 to 96 hours. PP three and PP four are mostly myoblasts. While muscle satellite cells are often seen mostly in PP five, the later prepl cells 96 hours or later PP six are regarded as adult stem cells and appear small, round and translucent.At the beginning of their isolation, they can be further characterized by immunohistochemistry for their expression of stem cell markers such as stem cell, antigen one and CDC 34. We've just show you how to isolate stem cell from a second mass of the mouse. When do this procedure, it's important to remember to maintain steroid during all the procedures.Once the stem cell has been isolated, make sure to culture those cells as low density to maintain the stem cell characteristics of the cells. So that's it. Thank you for watching and good luck with your experience And I wish you good luck to try to use this in your experiment.Thank you.

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