Mesenchymal Stem Cell Isolation: Techniques and Protocols" provides a detailed guide on the methods used to isolate mesenchymal stem cells, a key focus in regenerative medicine and tissue engineering. The article aims to assist researchers by clarifying these essential techniques.
What is Mesenchymal Stem Cell Isolation?
Mesenchymal stem cell isolation refers to the process of obtaining mesenchymal stem cells (MSCs) from various human tissues and organs. MSCs are considered primary adult stem cells with high proliferation capacity, wide differentiation potential, and immunosuppression properties, which make them unique for regenerative medicine and cell therapy.
The most widely studied sources of MSCs include bone marrow, adipose, muscle, peripheral blood, umbilical cord, placenta, fetal tissue, and amniotic fluid.The isolation of MSCs involves several steps, including:
- Tissue collection: The tissue containing MSCs is collected from the donor using a minimally invasive procedure
- Tissue processing: The collected tissue is processed to obtain a single-cell suspension
- Cell culture: The single-cell suspension is cultured in vitro under specific conditions to allow MSCs to adhere to the plastic surface of the culture dish
- Cell expansion: The adherent MSCs are expanded in vitro to obtain a sufficient number of cells for therapeutic applications
- Phenotypic characterization: The isolated MSCs are characterized by their expression of specific surface markers, such as CD73, CD90, and CD105
The methods of MSC isolation, morphological and phenotypic characterization, functional and differentiation properties, and therapeutic applications of MSCs are reviewed in detail in
Techniques for Isolation of Mesenchymal Stem Cells
There are several methods used for mesenchymal stem cell (MSC) isolation. These methods may vary depending on the tissue source and the specific research or clinical application. Here are some commonly used methods for MSC isolation:
- Adherent Culture Method: This method involves directly culturing the whole tissue or cell suspension obtained from the tissue source. MSCs have the ability to adhere to the plastic surface of the culture dish, allowing for their isolation and expansion.
- Density Gradient Centrifugation: This method involves layering the cell suspension onto a density gradient medium, such as Ficoll or Percol, and centrifuging it. The different cell populations separate based on their density, allowing for the isolation of MSCs from other cell types.
- Red Blood Cell Lysis: In some cases, such as when isolating MSCs from bone marrow, red blood cell lysis may be performed to remove erythrocytes from the cell suspension. This step helps to enrich the population of MSCs.
- Magnetic-Activated Cell Sorting (MACS): MACS is a technique that utilizes magnetic beads conjugated with specific antibodies to selectively bind and isolate MSCs based on their surface markers. This method allows for highly specific and efficient isolation of MSCs.
- Flow Cytometry Sorting: Flow cytometry can be used to sort and isolate MSCs based on their specific surface markers. This method allows for the isolation of pure populations of MSCs with high precision.
It is important to note that the choice of method may depend on factors such as the tissue source, the desired purity of MSCs, and the intended application. Researchers and clinicians often optimize and adapt these methods to suit their specific needs.
Enzymatic digestion technique
The enzymatic digestion technique is a widely used method for isolating MSCs. In this method, tissue samples undergo enzymatic digestion using enzymes such as collagenase to disaggregate the extracellular matrix, thus liberating the MSCs within the tissue.
Density gradient centrifugation technique
Following enzymatic digestion, the cell suspension is usually subjected to density gradient centrifugation. This technique separates cells based on their density, allowing lighter cells (like MSCs) to be isolated from the heavier cells and debris.
Selective adhesion technique
In the selective adhesion technique, the cell suspension is plated onto tissue culture surfaces. MSCs, being adherent cells, will attach to the surface, allowing for their selective separation from non-adherent cells.
Protocol for Isolation of MSCs from Bone Marrow
Materials required
Materials required for this protocol include a bone marrow aspirate, phosphate-buffered saline (PBS), heparin, 70% ethanol, 0.1% collagenase solution, and culture medium with necessary supplements.
Processing of bone marrow
The bone marrow aspirate is diluted with an equal volume of PBS and heparin to prevent clotting. The mixture is thoroughly mixed and layered onto a density gradient medium, followed by centrifugation.
Enzymatic digestion of bone marrow
The interphase layer containing mononuclear cells is collected and treated with collagenase to disaggregate clusters of cells. The mixture is incubated at 37 degrees Celsius until a single cell suspension is achieved.
Filtering and isolation of MSCs
The cell suspension is passed through a cell strainer to remove aggregates, followed by further centrifugation to pellet the cells. The cell pellet is then re-suspended in culture medium and plated onto a culture dish for further cultivation.
Protocol for Isolation of MSCs from Adipose Tissue
Materials required
Materials necessary include adipose tissue sample, PBS, collagenase solution, and culture medium with necessary supplements.
Processing of adipose tissue
The adipose tissue is minced into small pieces and washed with PBS to remove blood cells. Collagenase solution is added, and the mixture is incubated at 37 degrees Celsius.
Enzymatic digestion of adipose tissue
Enzymatic digestion breaks down the adipose tissue and releases the MSCs. The digestion is halted by adding culture medium.
Separation and isolation of MSCs
Subsequently, the cell suspension is centrifuged, and the supernatant (containing fat cells) is discarded. The pellet containing MSCs is re-suspended in fresh culture medium and plated onto a culture dish.
Protocol for Isolation of MSCs from Umbilical Cord
Materials required
Required materials include fresh umbilical cord, PBS, flushing medium, collagenase solution, and culture medium.
Preparation of umbilical cord
The umbilical cord is washed with PBS and cut into small pieces. The vessels are flushed with flushing medium to remove blood cells.
Enzymatic digestion of umbilical cord
Flushing medium containing collagenase is added to the pieces of umbilical cord and then incubated to allow for digestion.
Isolation and extraction of MSCs
The digested material is then centrifuged, and the resulting cell pellet is re-suspended in culture medium and plated onto a culture dish for further processing.
Protocol for Isolation of MSCs from Dental Pulp
Materials needed
Materials required include extracted tooth, PBS, enzyme solution, and culture medium with necessary supplements.
Processing of dental pulp
The tooth is split open, and dental pulp is separated. The pulp is then minced into tiny fragments, followed by enzymatic digestion.
Enzymatic digestion of dental pulp
Pieces of the pulp are incubated with enzyme solution to liberate MSCs entrapped within the tissue.
Isolation of MSCs
Post-digestion, the mixture is centrifuged, and the resulting cell pellet is re-suspended in culture medium. The suspension is then plated onto a culture dish for subsequent steps.
Culture and Expansion of MSCs
Materials necessary for culturing MSCs
Materials needed for culturing MSCs include culture dish, culture medium with necessary supplements, and a suitable incubator maintaining optimum temperature and CO2 levels.
Ideal conditions and media for MSCs growth and expansion
MSCs require a humidified environment at 37 degrees Celsius with 5% CO2. The culture medium must be the one that supports MSC growth and differentiation, usually containing necessary growth factors and antibiotics.
Passaging technique for MSCs
When MSCs reach 80-90% confluency, they should be passaged or sub-cultured. Cells are detached using an enzyme (like trypsin), re-suspended in fresh culture medium, and plated onto a fresh culture dish for continued growth.
Verification of MSCs After Isolation
Flow cytometry for MSCs
MSCs are characterized by their expression of specific surface markers. Flow cytometry is a technique used to analyze these markers and confirm the identity and purity of the isolated cells.
In vitro differentiation of MSCs
In vitro differentiation serves to confirm the multi-lineage potential of the isolated MSCs. Under specific culture conditions, MSCs can be induced to differentiate into adipocytes, osteocytes, or chondrocytes, among others.
Immunocytochemistry for MSCs
Immunocytochemistry allows visualizing specific markers of MSCs and assessing their potential for differentiation. It is performed using specific antibodies against the cellular markers of interest, followed by visualization under a microscope.
Preservation and Storage of Isolated MSCs
Freezing storage
For long-term storage, MSCs are cryopreserved at ultra-low temperatures. Cryoprotective agents like dimethyl sulfoxide (DMSO) are used to protect the cells from damage during the freezing process.
Thawing procedure
The thawing process should be quick to prevent the formation of ice crystals. Post-thaw, cells are transferred to pre-warmed culture medium, centrifuged to remove DMSO, and subsequently seeded in new dishes for culture.
Viability check post-storage
Cells viability post-thawing is usually checked by using viability dyes. Furthermore, the functionality of these cells should be assessed by passaging and differentiation assays to ensure they retained their characteristic properties after cryopreservation.
References
(1) Zhang W, Zhang F, Shi H, Tan R, Han S, Ye G, Pan S, Sun F, Liu X. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One. 2014 Feb 18;9(2):e88794. doi: 10.1371/journal.pone.0088794. PMID: 24558428; PMCID: PMC3928292.
(2) Tocci A, Forte L. Mesenchymal stem cell: use and perspectives. Hematol J. 2003;4(2):92-6. doi: 10.1038/sj.thj.6200232. PMID: 12750726.
