Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34+ cells to dimethyl sulphoxide

doi:10.1016/S0011-2240(02)00180-3

Cryobiology

Volume 46, Issue 1, February 2003, Pages 61-75

https://doi.org/10.1016/S0011-2240(02)00180-3 Get rights and content

Abstract

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34⁺ cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34⁺ cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34⁺ cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 °C. Values for the hydraulic conductivity were 3.2×10⁻⁸ and 2.8×10⁻⁷ cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2×10⁻⁷ and 7.4×10⁻⁶ cm/s, respectively. Clonogenic assays indicated that the ability of CD34⁺ cells to grow and differentiate was significantly impaired outside the limits 0.6–4× isotonic. Based on the Boyle van’t Hoff plot, the tolerable limits for cell volume excursion were therefore 45–140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 °C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34⁺ cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.

Section snippets

Collection of UCB donations

Cells were isolated from low-volume, UCB donations that would otherwise have been discarded and for which consent for research had been obtained. Each cord blood sample was collected from a single placenta into CPD according to London Cord Blood Bank (LCBB) protocols and stored at room temperature [24]. Storage times varied up to a maximum of 24 h post-donation. Six UCB donations were used for each set of experiments.

Cell isolation

Depending on the assay method used, either a mononuclear cell fraction

Osmotically inactive volume

CD34⁺ cells purified from UCB responded to anisotonic solutions by exhibiting an ideal osmometric response over the range 160–1800 mOsmol/kg (Fig. 1). Cell volume after equilibration in a 140-milliosmolal solution appeared to deviate slightly from ideal behaviour, however there was no significant difference between the intercept on the y-axis with and without the inclusion of this data point. Extrapolation of the regression line to infinite osmolality gave an osmotically inactive volume of

Discussion

The development of cryopreservation protocols for UCB stem cells has relied largely on an empirical approach utilising methods adapted from bone marrow and peripheral blood stem cells [35]. Studies of UCB stem cells have largely been directed to establishing that such methods provide an adequate level of post-thaw survival rather than attempting to derive optimal cryopreservation protocols from first principles [12], [27], [34]. A methodological approach has been advocated for cord blood and

Acknowledgements

We would like to thank the donor co-ordinators and processing staff of the London Cord Blood Bank for their help in providing cord blood. We are also indebted to the staff of the H&I Department NBS Colindale for their help and advice.

References (46)

T.A Amos et al.
Sources of human haematopoietic stem cells for transplantation—a review
Cell Transplant.
(1995)
F.G Arnaud et al.
Permeation of glycerol and propane-1,2-diol into human platelets
Cryobiology
(1990)
D.Y Gao et al.
Fundamental cryobiology of human hematopoietic progenitor cells. 1: Osmotic characteristics and volume distribution
Cryobiology
(1998)
A Hubel et al.
Cryobiophysical characteristics of genetically modified hematopoietic progenitor cells
Cryobiology
(1999)
O Kedem et al.
Thermodynamic analysis of the permeability of biological membranes to non-electrolytes
Biochim. Biophys. Acta
(1958)
F.W Kleinhans
Membrane permeability modelling: Kedem–Katchalsky vs a two-parameter formalism
Cryobiology
(1998)
D.S Krause et al.
CD34: structure, biology, and clinical utility
Blood
(1996)
L.E McGann et al.
Water permeability of human hematopoietic stem cells
Cryobiology
(1987)
D.E Pegg
Viability assays for preserved cells, tissues and organs
Cryobiology
(1989)
C.J Toupin et al.
Permeability of human granulocytes to water: rectification of osmotic flow
Cryobiology
(1989)

J.E Wagner et al.

Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment

Blood

(1992)

J.P Acker et al.

Comparison of optical measurement and electrical measurement techniques for the study of osmotic responses of cell suspensions

Cryo-Lett.

(1999)

S Armitage

Collection and processing of cord blood units

W.J Armitage et al.

The effects of osmotic stress on human platelets

J. Cell. Physiol.

(1985)

W.J Armitage et al.

Osmotic response of mammalian cells: effects of permeating cryoprotectants on nonsolvent volume

J. Cell. Physiol.

(1996)

P.G Beatty et al.

Impact of racial genetic polymorphism on the probability of finding an HLA-matched donor

Transplantation

(1985)

F Beaujean et al.

A simple, efficient washing procedure for cryopreserved human hematopoietic stem cells prior to re-infusion

Bone Marrow Transplant.

(1991)

E.A De Wynter et al.

Comparison of purity and enrichment of CD34⁺ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems

Stem Cells

(1995)

P Denning-Kendall et al.

Optimal processing of human umbilical cord blood for clinical banking

Exp. Hematol.

(1996)

D.A.T Dick

Cell Water

(1996)

C Donaldson et al.

Optimal cryopreservation of human umbilical cord blood

Bone Marrow Transplant.

(1996)

P.R Duggan et al.

Predictive factors for long-term engraftment of autologous blood stem cells

Bone Marrow Transplant.

(2000)

E Gluckman et al.

Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling

N. Engl. J. Med.

(1989)

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^☆: This work was funded by the Joely Bear Appeal.

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Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34+ cells to dimethyl sulphoxide☆

Abstract

Section snippets

Collection of UCB donations

Cell isolation

Osmotically inactive volume

Discussion

Acknowledgements

Cell Transplant.

Cryobiology

Cryobiology

Cryobiology

Biochim. Biophys. Acta

Cryobiology

Blood

Cryobiology

Cryobiology

Cryobiology

Blood

Comparison of optical measurement and electrical measurement techniques for the study of osmotic responses of cell suspensions

Cryo-Lett.

Collection and processing of cord blood units

The effects of osmotic stress on human platelets

J. Cell. Physiol.

Osmotic response of mammalian cells: effects of permeating cryoprotectants on nonsolvent volume

J. Cell. Physiol.

Impact of racial genetic polymorphism on the probability of finding an HLA-matched donor

Transplantation

A simple, efficient washing procedure for cryopreserved human hematopoietic stem cells prior to re-infusion

Bone Marrow Transplant.

Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems

Stem Cells

Optimal processing of human umbilical cord blood for clinical banking

Exp. Hematol.

Cell Water

Optimal cryopreservation of human umbilical cord blood

Bone Marrow Transplant.

Predictive factors for long-term engraftment of autologous blood stem cells

Bone Marrow Transplant.

Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling

N. Engl. J. Med.

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34⁺ cells to dimethyl sulphoxide☆

Comparison of purity and enrichment of CD34⁺ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems