The source from which SCs are obtained mostly depends on the disease to be treated which in turn reflects on the method of SC collection. Therefore every source has its unique method of SC collection:
From Bone Marrow
BM aspiration is the method of choice for the collection of different types of stem cells from the bone marrow. In this method, physicians use a special bone marrow aspiration kit that enables them to access the bone marrow. Accordingly, doctors use different sites and methods to aspirate bone marrow and collect SCs. The most commonly used site for collecting a bone marrow sample from adult humans is the iliac crest. A common method to obtain the sample is by using syringes coated with anticoagulants such as heparin or ACD. The syringes have to be pre-coated partially with anticoagulant prior to bone marrow aspiration. Then, the aspirated BM is transferred to a blood bag, also pre-coated and partially filled with the same type of anticoagulant used earlier to prevent clotting. After complete conveyance of all aspirated bone marrow, the sample is then diluted and filtered to remove bone fragments, cell clumps and fat. The marrow is subsequently centrifuged and processed further according to specific protocols.
The most appropriate and suitable method for the collection of SCs, based on their physical properties, is exemplified by the use of density gradient centrifugation (DGC), which is a widely utilized method worldwide. In this method, scientists usually use a medium with a specific density of 1.077g/ml such as Ficoll® to separate mononuclear cells from other cell types. Bone marrow blood sample is carefully layered over Ficoll® and then the tube is centrifuged at a certain speed with extra care not to mix the layers. Mononuclear cells usually appear as a white ring (buffy coat) in the tube after the completion of the centrifugation process.
This method allows the separation of bone marrow components according to specific densities of different cell types. The layer above the Ficoll layer that contains mononuclear cells (MNCs) is carefully aspirated and washed to remove remnant Ficoll and platelets. Stem cells are part of the MNC layer. Eventually, SCs obtained can be purified and used for ex vivo studies.
Methods for collecting SCs from BM include the use of leukapheresis machines that separate MNCs from a sample of blood in an automated and clinical-grade closed system.
From Peripheral Blood
Peripheral blood stem cells (PBSCs) can be collected using various procedures and methods. The most commonly used method is by leukapheresis machines that permit the separation of PB components automatically and in a closed system.
Leukapheresis allows the separation of PB mononuclear cells (PBMNCs) that contains a small percentage of stem cells in addition to agranulocytes (WBCs that do not contain granules). This fraction includes monocytes, lymphocytes, and small percentages of SCs.
To achieve an increase in the percentage of SCs in PB, physicians usually inject donors with an agent that stimulates hematopoiesis (blood cell production in the BM), promoting the mobilization of newly produced blood cells and SCs into the circulation. The most commonly used agent is Granulocyte-Colony Stimulating Factor (G-CSF). The donor is injected with G-CSF daily for 4-5 days at a dose of 10 µg G-CSF/kg weight of patient. This should be accompanied by monitoring the increase of WBC counts in the circulation and the percentage of CD34+ stem cells in the PB.
From Cord Blood
Cord Blood is collected directly after birth from the baby’s umbilical cord. The collection is done using a closed system by inserting a sterile needle into the umbilical vein and draining the blood into a collection bag. The sample is subsequently purified for SCs and unwanted cells are removed. The purified SC sample can be either used for treatment or it can be cryopreserved and stored in liquid nitrogen containers where the temperature can be as low as -196oC. The samples can be stored for several years and used at a later stage.
From Adipose Tissue (Fat)
Adipose tissue can be obtained by a liposuction procedure of certain areas of the body. The adipose tissue is subsequently processed and broken down in the lab using several procedures to isolate the stem cell fraction.