Progress in Cryopreservation of Stem Cells and Immune Cells for Cytotherapy

Cellular therapy with stem and immune cells has demonstrated significant success both in clinical treatments and the industrial market. Cryopreservation is a necessary and essential component of cellular therapy. In this chapter, first of all, some basic theories of cryoinjury and techniques in cryopreservation are reviewed. Then it focuses on the progress of cryopreservation of stem cells and immune cells, including new protocols and techniques, alternative cryoprotective agents (CPA), side effects after transplantation, and advances in reducing adverse reactions. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the CPA concentration or using alternative CPAs for cryopreservation, and removing CPA prior to infusion. Traditional and newly developed approaches including methods and devices for CPA removal are discussed. Future work is recommended including further optimization of cryopreservation protocols especially for lymphocytes; standardization of the optimized protocols with temperature monitoring and quality control; exploration of DMSO-free, serum-free, and even xeno-free media for cryopreservation; development of simple, reliable, and cost-effective devices for cryopreservation; and more fundamental cryobiological studies to avoid cellular injury.Keywords: cryopreservation, stem cell, immune cell, cytotherap

Correlation of infused CD3+CD8+ cells with single-donor dominance after double-unit cord blood transplantation.

Single-donor dominance is observed in the majority of patients following double-unit cord blood transplantation (dCBT); however, the biological basis for this outcome is poorly understood. To investigate the possible influence of specific cell lineages on dominance in dCBT, flow cytometry assessment for CD34(+), CD14(+), CD20(+), CD3(-)CD56(+), CD3(+)CD56(+) (natural killer), and T cell subsets (CD4(+), CD8(+), memory, naïve, and regulatory) was performed on individual units. Subsets were calculated as infused viable cells per kilogram of recipient actual weight. Sixty patients who underwent dCBT were included in the final analysis. Higher CD3(+) cell dose was statistically concordant with the dominant unit in 72% of cases (P = .0006). Further T cell subset analyses showed that dominance was correlated more with the naive CD8(+) cell subset (71% concordance; P = .009) than with the naive CD4(+) cell subset (61% concordance; P = .19). These data indicate that a greater total CD3(+) cell dose, particularly of naïve CD3(+)CD8(+) T cells, may play an important role in determining single-donor dominance after dCBT

Intracellular Disposition of Fludarabine Triphosphate in Human Natural Killer Cells

Purpose. Fludarabine is a key component of several reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Shortly after reduced-intensity conditioning, the percent of donor natural killer (NK) cells has been associated with progression-free survival. Insufficient suppression of the recipient’s NK cells by fludarabine may lead to lower donor chimerism; however, the effect of fludarabine upon NK cells is poorly understood. Thus, in purified human NK cells we evaluated the uptake and activation of fludarabine to its active metabolite, fludarabine triphosphate (F-ara-ATP), and assessed the degree of interindividual variability in F-ara-ATP accumulation. Methods. Intracellular F-ara-ATP was measured in purified NK cells isolated from healthy volunteers (n = 6) after ex vivo exposure to fludarabine. Gene expression levels of the relevant transporters and enzymes involved in fludarabine uptake and activation were also measured in these cells. Results. F-ara-ATP accumulation (mean ± s.d.) was 6.00 ± 3.67 pmol/1x106 cells/4 hours, comparable to average levels previously observed in CD4+ and CD8+ T-lymphocytes. We observed considerable variability in F-ara-ATP accumulation and mRNA expression of transporters and enzymes relevant to F-ara-ATP accumulation in NK cells from different healthy volunteers. Conclusions. Human NK cells have the ability to form F-ara-ATP intracellularly and large interindividual variability was observed in healthy volunteers. Further studies are needed to evaluate whether F-ara-ATP accumulation in NK cells are associated with apoptosis and clinical outcomes

Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution

Delayed myeloid engraftment after cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant–related morbidity and mortality. New culture strategies that increase the number of cord blood progenitors capable of rapid myeloid engraftment after CBT would allow more widespread use of this stem cell source for transplantation. Here we report the development of a clinically relevant Notch-mediated ex vivo expansion system for human CD34+ cord blood progenitors that results in a marked increase in the absolute number of stem/progenitor cells, including those capable of enhanced repopulation in the marrow of immunodeficient nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice. Furthermore, when cord blood progenitors expanded ex vivo in the presence of Notch ligand were infused in a clinical setting after a myeloablative preparative regimen for stem cell transplantation, the time to neutrophil recovery was substantially shortened. To our knowledge, this is the first instance of rapid engraftment derived from ex vivo expanded stem/progenitor cells in humans

GENE THERAPY CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy

The introduction of an inducible suicide gene has been proposed as a strategy to exploit the antitumor reactivity of donor T cells after allogeneic hematopoietic stem cell transplantation but permit control of graft-versus-host disease. However, there are several obstacles to this approach that may impair the ability of T cells to function and survive in vivo. These include the requirement for in vitro activation or long-term culture to introduce the transgene and obtain therapeutic cell numbers, the toxicity of drug selection to enrich transduced cells, and the immunogenicity of the transgene-encoded prod