Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium.

INTRODUCTION: Human fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me(2)SO concentration during cryopreservation of HFL hematopoietic cell preparations. METHODS: Human fetal liver (HFL) cells of 8-12 weeks of gestation were cryopreserved with a cooling rate of 1 degrees C/min down to -80 degrees C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me(2)SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose. RESULTS: The addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me(2)SO/0.3M sucrose mixture was comparable to cryopreservation in 5% Me(2)SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34(+) cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells. CONCLUSION: The inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me(2)SO, replacing serum and increasing the efficiency of cryopreservation.

The osmotic characteristics of human fetal liver-derived hematopoietic stem cell candidates.

Hematopoietic stem cells derived from fetal liver have promising therapeutic potential for allotransplantation but require a specific protocol to minimize the damage produced by cryopreservation procedures. In this study, a fundamental approach was applied for designing a cell preservation protocol. To this end, the biophysical characteristics that describe the osmotic reaction of CD34(+)CD38(-) human fetal liver stem cell candidates were studied using fluorescent microscopy. The osmotically inactive volume of the stem cell candidates was determined as 48% of the isotonic volume. The permeability coefficients for water and Me(2)SO were determined at T = +22 degree C: L(p) = 0.27 +/- 0.03 microm x min(-1)atm(-1), P(Me(2)SO)) = 2.09 +/- 0.85 x 10 (-4) cm x min(-1), sigma (Me(2)SO)) = 0.63 +/- 0.03 and at T = +12 degree C: L(p) = 0.15 +/-0.02 microm x min(-1)atm(-1), P(Me(2)SO)) = 6.44 +/-1.42 x 10 (-5) cm x min(-1), sigma (Me(2)SO)) = 0.46 +/- 0.05. The results obtained suggest that post-hypertonic and hypotonic stress are the possible reasons for damage to a CD34(+)CD38(-) cell during the cryopreservation procedure.

In utero stem cell transplantation: a European overview.

In utero stem cell transplantation (IUTx) is a therapeutic option for some lymphohaematopoietic disorders which can be diagnosed early in pregnancy. However, the promise of effective treatment before potentially life-threatening pathology becomes manifested has not been realised in all cases. This has prompted investigators to reassess their understanding of fetal haematopoiesis and of developmental pathways in the fetal immune system, to unravel the problem of failure to engraft after IUTx. The European Network for Fetal Transplantation (ENFET) was established to address these issues and to develop a concerted approach to IUTx. In this way, ENFET aims to study the basic biology of stem cell development and to put the findings into the context of engraftment potential following IUTx. One issue which has gained currency since ENFET was established, is the concept of stem cell 'plasticity', which has changed our view of the utility of IUTx so that there is now a real prospect for using this therapy in a larger range of disorders than was initially envisaged. Paradoxically, our current knowledge of stem cell plasticity must also change our views on the utility of IUTx for haematopoietic disorders, since it might offer some insights into the reasons for lack of engraftment observed in some of these conditions. The issue of in utero gene therapy is also being addressed by members of the ENFET consortium. Recent setbacks in postnatal gene therapy trials have put this therapeutic modality under intense scrutiny and there is much work still to be done before such therapy can realistically be offered in utero.