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Human Molecular Genetics 2008 17(R1):R48-R53; doi:10.1093/hmg/ddn079
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

This article appears in the following Human Molecular Genetics issue: Stem Cells and Regeneration [View the issue table of contents]

Good manufacturing practice and clinical-grade human embryonic stem cell lines

Christian Unger1,{dagger}, Heli Skottman4,{dagger}, Pontus Blomberg3, M. Sirac Dilber1 and Outi Hovatta2,4,*

1 Department of Medicine and 2 Department of Clinical Science, Intervention and Technology, Karolinska Institutet and 3 Vecura, Clinical Research Center, Karolinska University Hospital Huddinge, SE-14186 Stockholm, Sweden 4 Regea, Institute of Regenerative Medicine, University of Tampere and Tampere University Hospital, FI-33014 Tampere, Finland

* To whom correspondence should be addressed. Tel: +46 858583858; Fax: +46 858587575; Email: outi.hovatta{at}ki.se

Received January 30, 2008; Revised February 12, 2008; Accepted March 6, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 REFERENCES
 
Human embryonic stem cell (hESC) lines, after directed differentiation, hold the greatest potential for cell transplantation treatment in many severe diseases. Good manufacturing practice (GMP) quality, defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical-grade cells, offering optimal defined quality and safety in cell transplantation. Using animal substance-free culture media, feeder cells or feeder-free matrix in derivation, passaging, expansion and cryopreservation procedures, immune reactions against animal proteins in the cells, and infection risk caused by animal microbes can be avoided. It is also possible to apply GMP to animal components if no better options are available. In recent production of GMP-quality hESC lines, feeder cells had been cultured in fetal bovine serum, and the medium supplemented with an animal protein containing a serum replacement component. Using embryos cultured in a GMP laboratory, isolating the inner cell mass mechanically, deriving lines on human feeder cells originally cultured in xeno-free medium in a GMP laboratory, and using xeno-free media for derivation and culture of hESC lines themselves, GMP-quality xeno-free hESC lines could be established today. Human serum is a xeno-free component available today, but many chemically defined media are under development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
REFERENCES
 
There is a growing possibility of developing cellular therapies based on human embryonic stem cell (hESC) lines for the treatment of several malignant and non-malignant diseases. Given the rapid development in this field, clinical application of hESCs is expected in the near future. However, in order to provide cells with defined quality characteristics that are safe for the patient, good manufacturing practice (GMP) needs to be employed.

GMP in cell therapy
GMP is a quality assurance system used in the pharmaceutical industry. It ensures that the end product meets preset specifications. GMP covers both manufacturing and testing of the final product. It requires traceability of raw materials and also that production follows validated standard operating procedures (SOPs). In Europe, the requirement for cell therapy products is outlined in several directives and guidelines that are pertinent as regards hESCs (Directive 2004/23/EC, Commission Directives 2006/17/EC and 2006/86/EC). These set standards of quality and safety for donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Nevertheless, the legislation is still under development and new directives and guidelines are being produced (EU Regulation 1394/2007, guideline EMEA/CHMP/410869). The Regulation sets out tailor-made technical requirements and establishes new standards for clinical trials in the development of advanced medicinal products. The guideline will address development, manufacturing and quality control as well as non-clinical and clinical development of cell-based medicinal products.

In the USA, the Food and Drug Administration (FDA) has issued guidance in the form of Draft Guidance for Reviewers: Instructions and Template for Chemistry, Manufacturing, and Control (CMC) Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications (INDs) - 8/15/2003 and in the 21 CFR Part 1270, and 21 CFR Part 1271 regulations.

A common misconception is that clinical-grade hESCs may be produced by transfer of current methodology into clean room facilities. Such facilities are indeed important in avoiding microbial contamination of the product, but equally and perhaps even more important in implementing GMP will be the development of validated SOPs for the entire process, from cell isolation to freezing and storage of the cells. Another aspect of transfer of cell production to GMP standards is establishment of quality control methodology and release criteria of the cells produced. This will be challenging given the lack of European Medicines Agency-licensed cell therapy products in Europe and the fact that in the USA the FDA has not yet approved any human cell therapy product for sale. It should also be emphasized that implementing GMP will not necessarily ensure that the produced cells are of the highest possible quality or are the most efficient cells for a certain application. However, the benefit of GMP lies in the fact that the cells are produced in a reproducible manner and meet preset specifications that will ensure the safety of the patient.

Current hESC culture conditions
In hESC culture, there are several components that need to be developed according to GMP standards. The procedure begins with culture of the embryo in the in vitro fertilization laboratory, followed by isolation of the inner cell mass (ICM) of the blastocyst, and passaging of the cells. The feeder cells or the culture matrix have to meet certain standards. All the components of the culture and cryopreservation media, and all the processes involved, have to be described and validated according to the GMP quality system. GMP is a requirement for good clinical practice, a guideline for investigational products tested in clinical trials (CPMP/ICH/135/95).

Derivation
Establishment of the first permanent hESC lines was first described by Thomson et al. and by Reubinoff et al. (1,2). Since then, descriptions of the derivation of hundreds of new hESC lines (3) have been published. Most of the present lines have been derived from donated supernumerary blastocyst-stage embryos by isolation of the ICM using pronase and immunosurgery. However, immunosurgery may not be the optimal derivation method when poor quality discarded embryos with hardly visible ICMs are used (4). In addition, immunosurgery involves animal-derived substances—mouse antibodies and guinea pig complement—that are not desirable when considering xeno-free culture systems and cell transplantation. For standardization of derivation methods, more simplified protocols would be appreciated. Tyrode's acid or mechanical opening for removal of the zona pellucida, instead of pronase, and mechanical isolation of the ICM is advantageous, since there is no contact between blastocyst and animal-derived pronase, antibodies and complement. Such a procedure has recently been adopted for establishment of new hESC lines by Genbacev et al. (5). Our group developed flexible metal needles for rapid removal of the zona pellucida and mechanical isolation of the ICM. We have derived several new hESC lines using them (6). In addition, hESC lines have also been derived from whole blastocysts without isolating the ICM (7,8), and from morula-stage embryos (9), as well as from blastomeres cultured together in groups on the same plate in co-culture with hESCs (10,11).

Culture media
At first, commonly used hESC culture media contained fetal bovine serum (FBS) (1,2), but later the use of human serum instead of FBS in hESC culture media (12) and in derivation of new hESC lines was described (8). Owing to the facts that serum is a complex mixture containing unknown compounds and that serum batches vary in their capability of maintaining hESCs at an undifferentiated stage, the replacement of serum with defined components would be optimal for GMP production of hESCs. To overcome serum-related problems, several groups have optimized serum-free culture conditions for hESC lines using serum replacement (Ko-SR, Invitrogen) (9,13–15). This conventional serum replacement with basic fibroblast growth factor (bFGF) is widely used. Although the use of Ko-SR in hESC culture medium provides more standardized and better defined culture conditions compared with serum-containing medium, it contains AlbuMAX, a lipid-rich albumin fraction of bovine serum and bovine transferrin, and is therefore not free of animal-derived components (16).

Most of the reported feeder-free culture methods involve the use of conditioned media from feeder cells. Some research groups have reported successful use of feeder-free culture conditions with non-conditioned Ko-SR containing hESC culture medium supplemented with activin A and bFGF (17), a combination of the bone morphogenetic proteins signaling antagonist Noggin and high concentrations of bFGF (18,19), and activin A, nicotinamide and keratinocyte growth factor (20). Important work has been carried out involving the replacement of xeno components in media with more defined media and supplements. Li et al. as well as Genbacev et al. used xeno-free X-VIVO 10 with bFGF (5,21), Vallier et al. (22) used chemically defined medium with activin A, nodal and bFGF and Ludwig et al. (23) used xeno-free defined culture medium with bFGF, LiCl, GABA, TGFb and pipeolic acid.

The next step towards entirely xeno-free derivation and maintenance of hESCs would be the combination of mechanical isolation of the ICM, use of human feeder cells derived and cultured in xeno-free medium, and use of xeno-free defined culture medium in hESC culture. Conventional Ko-SR medium contains animal-derived components, which makes it sub-optimal when considering pathogens and possible immunoreactions. This medium, however, was very recently used in the derivation of six clinical-grade hESC lines aimed at clinical applications (24). The lines were derived and propagated using a GMP formulation of Ko-SR, but it remains to be seen if the establishment and culture of the next clinical-grade hESC lines will be performed under GMP conditions without any trace of xeno material.

Feeder cells and feeder-free culture
Shortly after the advent of hESC lines, a need for more human adaptation was pushing the development of human feeder cells for hESC growth, turning away from the commonly used murine embryonic fibroblast feeder cells (25–27). This was mainly necessary to reduce the number of factors potentially contaminating valuable and unique hESC lines, reducing their clinical potential. Although the risk of transmitting murine viruses seems low (28), the potential immune rejection of xeno-proteins in hESCs, as demonstrated by Martin et al. (29), shows the need for a complete human cell culture system in order to achieve the full clinical potential of hESCs. Of course it would be most optimal to have a feeder-free, fully-defined system, but these are currently under development (23). However, until these systems are applicable for most hESC lines (30), appropriately screened and GMP-grade, human feeder cells represent the best possible support for clinical-grade hESC lines. In a recent publication by Crook et al. (24), the authors describe how GMP-grade hESC lines were successfully derived using GMP-grade human feeder cells grown in medium with GMP-quality FBS. In the light of data published by Martin et al. (29), these cells might potentially be rejected in clinical application as a result of incorporation of non-human sialic acid, against which many humans produce antibodies. Therefore, a xeno-free system is required for these cells.

Various human cell types have been used and they have been shown to support hESC growth and even their derivation. Fibroblasts supporting hESC lines have been derived from fetal skin and muscle of aborted embryos (12,31), and from adult tissues, such as fallopian tube and foreskin (25,26). Cheng et al. (32) used human marrow-derived stromal cells to expand hESCs and Lee et al. (33) used cells from uterine endometrium as feeders. Even hESC-derived fibroblast feeders have been used, avoiding involvement of other sources of tissue (34). However, in such cases a hESC line has to be derived first, which requires feeder or matrix support. We have applied human foreskin fibroblasts in hESC cultures (26) from the first derivations, and have since then derived many hESC lines, showing their applicability (35). As yet no GMP-grade xeno-free human feeder cells are available, although research work has been published in which human serum has been used instead of FBS (25).

Feeder-free derivation: Derivation of the first feeder-free hESC lines was described by Klimanskaya et al.(36), using a mouse-derived matrix. Even though such a matrix can be sterilized it is probably not optimal for clinical use. Ludwig et al. (23) derived two new hESC lines on Matrigel and human laminin in a chemically defined medium. One of the lines had the karyotype 47, XXY, which may have come from the embryo, but the other line also gained an extra chromosome at passage level 40. Feeder-free cultures may be so demanding for hESCs that they become more prone to abnormalities (37). Hence, using human GMP-quality feeder cells in xeno-free conditions is the safest method so far to derive clinical-grade hESCs.

Passaging and freezing
Passaging of the cells is a step in which the constituents also need to be GMP compatible. Mechanical passaging has been applied by many teams, and it does not involve any additional substances, making it an excellent method to be applied in a GMP system. However, enzymatic passaging is needed in many situations. In bioreactors, use of enzymes is easier (38). If flasks are used in cultures, enzymatic splitting is needed. GMP-quality human collagenase is commercially available (Serva, Germany) and was used in recent study (24), but commercially available trypsin preparations, including human recombinant trypsin, are not of GMP quality.

Freezing, cryostorage and thawing processes should also be of GMP standard, with attention to the cryoprotectant medium, protein source and quality of the liquid nitrogen. Contact with the liquid nitrogen should be avoided, and there should be contamination risk-free storage of the straws or vials. Both vitrification and conventional slow cooling/rapid warming have been successfully applied in cryostorage of hESCs, as reviewed by Hunt and Timmons (39). Previously used methods cannot be used clinically without modifications. Richards et al. (40) described a xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws and use of human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant. Long-term storage should be in the vapor phase of liquid nitrogen. This process may be transferred to GMP. Crook et al. (24) froze their clinical-grade cells using slow programmed freezing in a GMP-manufactured xeno-free protein-free medium in hermetically sealed straws. Instead of straws, Fujioka et al. (41) successfully cryopreserved primate ESCs by means of a vitrification method involving use of conventional cryovials, yielding a survival rate of ~6.5% for monkey ES cells and 12.2% for human ES cells. This system was not of GMP quality, but it might be possible to modify it to be compatible.

Clinical-grade hESC lines
Current situation
The safest option to obtain clinical-grade cells would be to derive the lines from the very beginning in GMP-compatible conditions. This would also require the embryos to have been produced and cultured in such conditions (24). According to new European Union Tissue Directives (EU directives 2004/23/EC, 2006/17/EC), all in vitro fertilization laboratory work should be carried out in conditions that facilitate obtaining donated embryos for stem cell line derivation. As an alternative, culture of existing lines in laboratories and culture systems meeting GMP requirements has been presented. This would require up to ten passages within such conditions, and after that extensive testing to show that the cells do not contain known pathogens and that they are chromosomally normal.

To establish clinical-grade hESC lines, Crook et al. (24) devised extensive standard operational procedures, validated protocols for ethics approval and consent, validated a good tissue culture practice system for embryo cultures, isolated the ICM mechanically, or plated whole blastocysts. The ICM was plated on commercially available GMP-quality foreskin feeder cells, which were not xeno-free because they had been established and propagated in FBS-containing medium. The FBS was of GMP quality. The six chromosomally normal hESC lines they produced meet clinical quality even though they are not xeno-free. The presence of known pathogens of human and animal origin was assessed in both the embryo donors and the cell lines. It remains to be seen if lines cultured using FBS feeders are rejected more easily than xeno-free lines.

Optimally, a chemically defined GMP-quality culture medium containing only human substances should be used. The feeders would have been cultured in similar conditions. Mechanical isolation of the ICM, and mechanical passaging would be optimal. If enzymes are used, they should be only GMP-quality human proteins. Feeder-free derivation and culture on a GMP-quality human extracellular matrix would be perfect, but such a system does not yet exist (Fig. 1).


Figure 1
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  		Figure 1. Foreseen steps towards clinical applicable hESC-derived cells. Overview showing current status with optimization possibilities and GMP-processing, plus the still missing future parts required to enter clinical trials.

 
Future directions
Generation of clinical-grade hESCs is an important first step towards a wide range of possible future treatments. However, clinical-grade cell lines are not the final product. From a regulatory point of view, it is at present not even clear whether hESC cells should be considered as active pharmaceutical ingredients, raw material or intermediate products. Once the cell lines have been established, the next phase will be setting up procedures for scale-up and expansion of the cells into a product that may be given to larger patient groups. To realize this, GMP adaptation of the subsequent steps, which involves induced differentiation towards other cell types, might be an even greater challenge (Fig. 1). According to the application area of the hESC-derived cells, re-evaluation of GMP-verified hESC culture conditions must be carried out to ensure that all safety precautions are taken. So far possible protocols for hESC culture (21) and differentiation (42) are published, but the complete FDA approval for a clinical trial needs to be awaited.

For optimal clinical quality, immunological problems other than those caused by xeno-components should be solved. To prevent immune rejection of hESC-derived tissue, a bank of many hESC lines will be required. Taylor et al. (43) estimated that 150 cell lines would be necessary and beneficial for much of the population. Somatic cell nuclear transfer might be an option in the future. Such ESC lines have been obtained in mice (44), but so far not in humans, even though cleavage-stage embryos and a blastocyst have been obtained (45–47).

Autologous cells would be beneficial in many indications involving cell transplantation. The new development of reprogramming somatic cells into highly potential hESCs (induced pluripotent stem cells), now also successful in humans, may in the future make this possible (48,49). Reprogramming is carried out by genetic modification with viral vectors and so far no such cell lines are in clinical use, owing to the dangers and ethical issues involved in current gene therapy protocols (50,51) (http://www.esgct.org). Therefore, and because of the danger connected with inserted genes causing tumor growth (52), this very recent development is far from the production of clinically applicable hESCs. However, future development will most likely lead to safer ways of reprogramming somatic cells and will involve all optimization data available as regards current hESC lines. Furthermore, clean human fibroblasts derived now as feeders by means of GMP methodology could later provide important start-up material for new hESC lines, applying future technologies.


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge funding from the Swedish Research Council, the Research and Development Funds of Stockholm County and Karolinska Intitutet, the Juvenile Diabetes Research Foundation International (JDRF), and the European Union (ESTOOLS) (to O.H. and S.D.), the Academy of Finland (to H.S. and O.H.), TEKES the Finnish Funding Agency for Technology and Innovation (to H.S. and O.H.), the Competitive research funding of the Pirkanmaa Hospital District (to H.S.).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} These authors contributed equally to this work. Back


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