Plasma clots gelled by different amounts of calcium for stem cell delivery.
OBJECTIVE
Freshly prepared autologous plasma clots may serve as a carrier matrix for expanded multipotent mesenchymal stromal cells (MSCs) or bone marrow cells. By varying the calcium concentration, plasma clots with different properties can be produced. The purpose of this in vitro study was to determine the optimal calcium concentrations for the clotting process, intra-clot cell viability, and clot lysis.


METHODS
Different plasma clots were prepared by adding an equal volume of RPMI1640 (with or without MSCs) to citrate plasma (either containing platelets or platelet-free). Clotting was initiated by the addition of CaCl(2) (10 g/100 ml H(2)O, 10 % solution). The final concentration of CaCl(2) ranged from 1 to 10 % by volume of plasma. Viability and distribution of the MSCs were analysed by calcein-AM/propidium iodide staining. MSC-embedded plasma clots were dissolved with trypsin (0.25 %), and recovered cells were further incubated for 1 week under cell culture conditions.


RESULTS
The viability of MSCs embedded in clots formed by the addition of 1-8 % by volume CaCl2 was not affected by incubation of up to 1 week. In contrast, clots produced by higher volumes of CaCl(2) solutions (9-10 % by volume of plasma) showed decreased numbers of viable cells. Intra-clot cell proliferation was highest in clots produced by addition of 5 % CaCl(2) by plasma volume. Osteocalcin release was not influenced in platelet-free plasma but decreased in platelet-containing plasma. Morphological analysis of stained recovered MSCs revealed that lysis of the plasma clot did not affect cell morphology or subsequent spontaneous proliferation.


CONCLUSIONS
Clot formation and clot stability can be controlled by changing the concentration of CaCl(2) added to plasma. The addition of 5 % CaCl(2) produced a plasma clot with optimal results for stem cell delivery.
Department of General and Trauma Surgery, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany. jangessmann@me.com
Jan
J
Gessmann
Dominik
D
Seybold
Elvira
E
Peter
Thomas Armin
TA
Schildhauer
Manfred
M
Köller
D001777:Blood Coagulation / Q000187:drug effects*; D016026:Bone Marrow Transplantation / Q000379:methods*; D002122:Calcium Chloride / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D015675:Osteocalcin / Q000096:biosynthesis; D010949:Plasma
D015675:Osteocalcin; D002122:Calcium Chloride