Preparation of bone marrow for the cell separation process

Material: The separation of stem cells from a bone marrow (BM) is an in-vitro procedure, it is performed with bone marrow in blood bags. For this process all generations of Fresenius Blood Cell Separators can be used: AS104, AS.TEC 204 as well as COM.TEC (fig. 2). The devices of the three generations are different in the ease of handling, but the principle is similar.

For bone marrow stem cell (BMSC) collection the disposable P1Y, article number 9400421 (fig. 2) has to be used. To have a properly running separation the BM has to be transferred into the BMSC bag set (article number 90073419, fig 1). Each of the bags has a capacity of 2500 ml and has the correct connectors fitting to the P1Y set used for the BMSC separation.

2500 m l

2500 m l

Figure 1 - disposable BMSC

General information: Bone marrow is a soft tissue which is in the cavities of bones. It is composed of the red or hematopoietic marrow, and fat in different amounts. Proliferation and differentiation of the stem cells build up the different types of blood cells. The marrow is nerved by numerous large and small blood vessels, because the hematopoietic process needs a lot of nutrients for the highly active processes, and to transport the adult blood cells into the circulation. Since the oxygen and carbon dioxide are necessary for every single cell in the body and the immune system responsible for the repair and defense processes proliferation and differentiation takes place in enormous extent.

Page 1 of 3

1.

2.

3.

4. 5.

Preparation of the marrow for the separation process: The bone marrow is collected by puncture of the iliac crest of the donor/patient in the operation theatre. The syringes used contain heparin (sometimes ACD-A) for anticoagulation. The marrow aspirated contains bone particles and fat, and can not be used for BMSC separation without further preparation steps, which are usually performed in a sterile bench in a laboratory. First of all the aspirated BM has to be filtered to get rid of bone fragments, particles as well as blood clots. This is usually done with a filter with a mash size of 170-200 m. The filtrate is collected in bags. If the BM has a low hematocrit (< 27 %), it might be necessary to eliminate some plasma by a soft spin. With this soft spin the amount of fat is reduced: this fat would disturb the cell separation process, because it would stick within the separation chamber of the centrifuge causing pressure problems and a decrease in efficiency of the stem cell collection. If the hematocrit of the aspirated BM is high enough (~ 30%), a soft spin is not necessary. To eliminate fat from the BM it is suggested to leave it hanging until fat separates by gravity. Finally, a pre sample is taken before starting the sell separation to control the quality.

ATTENTION It is of high importance to eliminate particles and fat from BM before the separation is started: Fat affects the sedimentation of the cells within the separation chamber, and fat as well as bone particles, blood clots, and micro-aggregates might cause clogging the separation chamber of the P1Y disposable.

Page 2 of 3

Fat elimination: 2 possibilities: 1. Gravity: Leave it hanging until fat separates by gravity. Hang up the bag(s) until the fat collects on top of the suspension and press it out. 2. Centrifugation: a) Perform a soft spin and press the fat out. b) A soft spin might increase the hematocrit up to 70 - 80%, therefore, it should be diluted by 4% albumin solution to a convenient hematrocrit (~ 30%).

Figure 2 - Disposable P1Y

Figure 3 - Cellseparator COM.TEC

Page 3 of 3