Elsevier

Cytotherapy

Volume 12, Issue 3, 2010, Pages 338-348
Cytotherapy

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood

https://doi.org/10.3109/14653241003695034Get rights and content

Abstract

Background aims

The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion.

Methods

In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated.

Results

The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character.

Conclusions

Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

Introduction

The cord blood (CB)-derived unrestricted somatic stem cell (USSC) is a non-hematopoietic multipotent cell displaying mesodermal, endodermal and neural differentiation capacities in vitro and in vivo (1., 2., 3., 4.). Together with the ability to migrate and accumulate to sites of injury and its response to growth factors (5,6), USSC is a promising candidate for future cellular therapy and clinical application in allogeneic settings, as well as for reprogramming for induced pluripotent stem cells (iPS) (7., 8., 9., 10., 11.).

Compliant clinical generation of cell drugs requires adherence to strict regulations to achieve high-product quality for downstream applications. Good manufacturing practice (GMP) is an essential part of the quality management system to ensure product safety and effectiveness. According to current European law, cell-based medical products should be propagated under principles of GMP when they are used in phase I studies (12).

We describe GMP-compliant generation of USSC during the pre-clinical phase to ensure consistent production, reliability and quality prior to clinical use. To conduct pre-clinical development of USSC, mononucleated cell (MNC) isolation from fresh CB, clinical-scale expansion, cryopreservation and thawing of USSC were performed in compliance with GMP standards. Release criteria for the product were defined by assessment of the immunophenotype, potency and genetic stability. Evaluation of sterility, pyrogens and endotoxins led to high safety and quality standards of the final cryopreserved and thawed products.

Section snippets

Collection of fresh CB and the processing environment

CB samples were obtained from donors meeting the criteria for hematopoietic banking according to the latest regulatory guidelines in Europe (Table I). Donated CB for USSC generation was used only if the initial cell count was not sufficient for hematopoietic banking.

Umbilical CB was collected by vein puncture in a blood collection bag (MSC1206DU; Macopharma, Langen, Germany) containing 29 mL citrate phosphate dextrose. Subsequent processing took place within 48 h under GMP conditions according to

Results

GMP-conforming generation and cultivation of USSC is mandatory for clinical applications. To ensure safety and reproducibility, the automated Sepax cell-processing system (Biosafe) was used for MNC separation from 98 fresh CB samples, similar to a methodology described previously (13).

The GMP-conforming Sepax-mediated MNC isolation of 98 CB samples (mean volume 84.61 ± 17.02 mL, range 51–136 mL) resulted in a mean recovery of 42.37 ± 10.09% NC (range 16.13–67.55%) and 63.03 ± 14.57% MNC (range

Discussion

Since its use as a hematopoietic stem cell transplant for many indications, such as leukemia and lymphoma, umbilical CB is no longer considered as biologic waste. The application of CB for non-hematopoietic indications, especially in regenerative fields, has undergone much investigation in recent years: the discovery of non-hematopoietic stem cells in CB (1) was met enthusiastically and led to it becoming a promising candidate in the fields of regenerative medicine, clinical research and

Acknowledgments

We gratefully acknowledge the Deutsche Forschungsgemeinschaft (DFG) for funding the research group FOR 717, including the project Ko2119/6-1, and the German José Carreras Leukemia Foundation for supporting us with grant DJCLS-R07/05v. We would like to thank A. Lefort and D. Stapelkamp for their technical support.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References (44)

  • SpeesJ.L. et al.

    Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy

    Mol Ther

    (2004)
  • ShettyP. et al.

    Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

    Cell Biol Int

    (2007)
  • CarrancioS. et al.

    Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification

    Exp Hematol

    (2008)
  • MullerI. et al.

    Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

    Cytotherapy

    (2006)
  • MizunoN. et al.

    Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

    Cell Biol Int

    (2006)
  • StuteN. et al.

    Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use

    Exp Hematol

    (2004)
  • LiedtkeS. et al.

    Oct4 and its pseudogenes confuse stem cell research

    Cell Stem Cell

    (2007)
  • Fallahi-SichaniM. et al.

    In vitro differentiation of cord blood unrestricted somatic stem cells expressing dopamine-associated genes into neuron-like cells

    Cell Biol Int

    (2007)
  • KoglerG. et al.

    A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential

    J Exp Med

    (2004)
  • GreschatS. et al.

    Unrestricted somatic stem cells from human umbilical cord blood can be differentiated into neurons with a dopaminergic phenotype

    Stem Cells Dev

    (2008)
  • JagerM. et al.

    Bone healing and migration of cord blood-derived stem cells into a critical size femoral defect after xenotransplantation

    J Bone Miner Res

    (2007)
  • BuchheiserA. et al.

    Cord blood for tissue regeneration

    J Cell Biochem

    (2009)

    • Cited by (0)

    View full text
    Copyright © 2010 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.