Mr. Koller et al., CLINICAL-SCALE HUMAN UMBILICAL-CORD BLOOD-CELL EXPANSION IN A NOVEL AUTOMATED PERFUSION CULTURE SYSTEM, Bone marrow transplantation, 21(7), 1998, pp. 653-663
Use of umbilical cord blood (CB) for stem cell transplantation has a n
umber of advantages, but a major disadvantage is the relatively low ce
ll number available. Ex vivo cell expansion has been proposed to overc
ome this limitation, and this study therefore evaluated the use of per
fusion culture systems for CB cell expansion. CB was cryopreserved usi
ng standard methods and the thawed unpurified cells were used to initi
ate small-scale cultures supplemented with PIXY321, flt-3 ligand, and
erythropoietin in serum-containing medium. Twelve days of culture resu
lted in the optimal output from most CB samples. Frequent medium excha
nge led to significant increases in cell (93%), CFU-GM (82%) and LTC-I
C (350%) output as compared with unfed cultures. As the inoculum densi
ty was increased from 7.5 x 10(4) per cm(2) to 6.0 x 10(5) per cm(2),
the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM out
put reached a plateau at 6.0 x 10(5) per cm(2), whereas LTC-IC output
continued to increase up to 1.2 x 10(6) per cm(2). Because the increas
e in culture output did not increase linearly with increasing inoculum
density, expansion ratios were greatest at 1.5 x 10(5) per cm(2) for
cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent s
troma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-C
SF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-beta, TNF-alpha, and MIP-1 alpha
) that were also found in bone marrow (BM) cultures, with the exceptio
n of IL-11 (found only in BM) and IL-3 (found in neither). Culture out
put was remarkably consistent from 10 CB samples (coefficient of varia
tion 0.3) indicating that the procedure is robust and reproducible. Tw
o commercial serum-free media were evaluated and found to support only
approximately 25% of the culture output as compared with serum-contai
ning medium. Implementation of optimal conditions in the clinical scal
e, automated cell production system (CPS) showed that the process scal
ed-up well, generating 1.7 x 10(7) CFU-GM (298-fold expansion) from 1.
2 x 10(8) thawed viable nucleated CB cells (n = 3). The ability to gen
erate >10(7) CFU-GM from <15 mi of CB within this closed, automated sy
stem without the need for extensive cell manipulations will enable cli
nical studies to test the safety and efficacy of expanded CB cells in
the transplant setting.