Outline
Introduction 409
Basic Principles of Cell Banking 410
Donation 410
Collection 411
Processing 411
Storage 417
Selection of Cells for Transplantation and Delivery 418
Thawing and Viability Assessment 418
Information System 418
To date, stem cells from different sources have been used mainly for research purposes. Potential medical applications of stem cells involve inflammatory, neurodegenerative, metabolic and musculoskeletal diseases, and diseases of the heart and blood vessels. Future clinical applications will require a large number of cells and therefore the development of stem cells banks will be necessary. Cell banking is the preservation of a cell stock in the originally obtained state. These banks must ensure the availability, quality and safety of the stored cell products, especially when the stored cells are mainly for clinical use. The development of processing methods to generate cells in compliance with current good manufacturing practices is mandatory to ensure the quality of the cell products.
Introduction
Stem cell-based strategies have become a tool for a diverse range of medical applications such as organ and tissue repair, a complement to organ transplantation (mainly by modulating the immune response) and an alternative to organ transplantation .
Hematopoietic stem cells (HSCs) derived from bone marrow were the first adult stem cells used for therapy . Later, peripheral blood and cord blood were consolidated as alternative sources of HSCs for transplantation. The experience gained in hematopoietic progenitor banking for transplantation provides a model for general stem cell banking. Many cord blood banks, both private and public, have been established in recent years and play an important role throughout the world . To date, stem cells from different sources have been used mainly for research purposes. Potential medical applications of stem cells include inflammatory, neurodegenerative, musculoskeletal and metabolic diseases, and diseases of the heart and blood vessels .
The variety of cell lineages with different functions in the kidney makes it difficult to find the stem cell . However, most of the cells that reveal stemness conditions (ability to generate differentiated progeny of multiple cell types, ability to repopulate tissues in vivo and self-renewal) from different human tissues (e.g. bone marrow, adipose tissue, dental pulp and cord blood) show similar cell structure and, therefore, similar requirements for storage processing .
The possibility of re-creating in the laboratory a pluripotent state in stromal fibroblastic cells and other differentiated somatic cell types [e.g. induced pluripotent stem (iPS) cells] introduces the idea of personalized stem cell therapy and may lead to the development of substances for regenerative medicine . Nevertheless, much still needs to be learned regarding the behavior of iPS cells before they or their products can be used in the clinic .
Future clinical applications will require a large number of cells and therefore the development of stem cells banks will be necessary. Cell banking comprises the preservation of a cell stock in the originally obtained state. These banks must ensure the availability, quality and safety of the stored cell products, especially when the stored cells are mainly for clinical use. Critical issues involving stem cell banking are summarized in Table 26.1 .
| Collection area | Stem cell bank | Transplant center |
|---|---|---|
| Donor selection | Reception | Selection |
| Stem cell collection | Processing | Transplantation |
| Cryopreservation and storage | Follow-up | |
| Biological controls | ||
| Shipment to transplant center | ||
| Follow-up |
Basic Principles of Cell Banking
Stem cell banking for therapeutic use requires several standards to be maintained:
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Quality : development of processing methods to generate cells in compliance with current good manufacturing practices (GMP) to ensure the quality of the products. Commission Directive 2003/94/EC of October 2003 regulates the principles and guidelines of GMP with respect to medicinal products for human use and investigational products in Europe .
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Safety : applying strict measures to avoid the risk of disease transmission. Cellular therapy products are derived from human sources and, therefore, carry the risk of transmitting infectious agents.
- •
Efficiency : providing products that maintain the biological properties that are useful for human health (clinical perspective) and performing all the activities related to stem cell banking at a lower cost without reducing the quality.
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Traceability : allowing the ability to track specific information at every step in the process chain. This must be achieved confidentially.
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Transparency : offering accurate information to users and institutional organisms according to current concepts in medicine and regulations.
- •
Benchmarking : establishing indicators to compare the model with other ones and optimizing the services by implementing a continuing program of quality improvement.
To meet these issues and ensure reliable quality, the bank needs a quality assurance system of management. This system must comply with current professional standards and statutory legislation. Basic principles of GMP guidelines involve quality assurance, personnel, premises and equipment, documentation, production, quality control, complaints and product recalls, and self-inspection and quality audits . Moreover, the design of stem cell bank facilities must focus on guaranteeing the quality and safety of the cell products.
The personnel working in the bank must be familiar with the quality requirements, carrying out appropriate training programs, and must know their responsibilities (management, medical advisory, technical work, quality assurance). The standard operating procedures and associated documents of the cell bank must be included in a manual and be periodically reviewed to ensure their suitability.
The implementation of such a quality assurance system allows relationships to be established among cell banks for clinical use based on a common principle of applying standards that are as high as necessary, which is important to encourage cooperation at the international level.
By concentrating resources and expert knowledge, centralization is an efficient policy for cell and tissue banks at regional and national levels. Centralization is especially important for public non-profit banks. The international network of umbilical cord blood banks, intended to achieve the fast and efficient location of products according to human leukocyte antigen (HLA) compatibility criteria, is a good example of the implementation of common standards for international cooperation in stem cell banking. However, there continues to be a major effort to standardize banking and regulate all the steps to provide the highest quality for patient use. Organizations such as the American Association of Blood Banks, the American Red Cross, the American Society of Blood and Marrow Transplantation, the European Blood and Marrow Transplantation Society, Eurocord, the Foundation for the Accreditation of Hematopoietic Cell therapy, the International Society for Hematotherapy and Graft Engineering, the Joint Accreditation Committee of ISHAGE-Europe and EBMT, Netcord and the National Bone Marrow Donor program make sure that the quality and standards in cord blood banking are established and met .
Donation
Proper informed consent has to be obtained for the intended use (autologous and/or allogeneic administration) as well as for retrieval, processing, testing, storage and data management. In addition, the possibility of alternative use in research must be included when cells are not used for therapeutic purposes. Other possibilities could be a change from the autologous to allogeneic condition, once autologous use has been completely discarded and when donor selection criteria are in compliance. In any case, the donor must be informed and advised in a comprehensive manner according to the scientific and clinical evidence.
Donor selection criteria are based on an assessment of medical and behavioral history. Potential donors must answer a questionnaire specially designed to identify specific risk factors for hepatitis B and C, human immunodeficiency virus (HIV), syphilis and other infectious diseases. This is the first step in donor screening and the most important one in order to avoid the risk of transmissible disease. The exact questions to be asked, the information to be obtained and the source of that information have to be regularly reviewed and updated by specialized professionals belonging to accredited scientific societies, international health institutions and government institutions (American Association of Tissue Banks, American Association of Blood Banks, International Society for Cell Therapy, European Association of Tissue Banks, Food and Drug Administration, European Medicines Agency, etc.). The kind of stem cells to be collected should also be covered in the questionnaire .
The second step taken to minimize the risk of infection is testing donor blood samples for specific infectious markers (hepatitis B and C, HIV, syphilis, etc.) . The decision on the diseases to be screened depends mainly on the origin of the donor and also on the geographical scope of the bank’s services (different countries have distinct requirements). Epidemiological institutions offer current information on this issue. Samples need to be stored for additional testing, especially in the case of products that can be stored for long periods. Donor samples must be tested in laboratories accredited by international societies.
It is important to take into account that cell manipulation proceedings usually start before the donor’s test results are known. As biological products of human origin, tissues, cells and associated samples are considered to be risk materials for disease transmission, and protective measures must be implemented during manipulation. In addition, the risk of contamination in the opposite direction (from the operator and the environment to the biological products) has to be avoided. This concern will be present in all of the following procedures.
Collection
Cells and tissues are usually collected under operating theater conditions. An aseptic environment is fundamental to minimizing the risk of contamination. If cells became contaminated at this step, this condition will be maintained in the following phases of processing, because of the difficulty in applying sterilization procedures which may irreversibly damage cells . For instance, microbiological contamination can lead to 1–15% of cord blood being discarded .
Cells are collected as a suspension (e.g. hematopoietic progenitors) or included in tissue fragments (e.g. adipose tissue). In either case, specific environmental conditions (container, temperature, transport solution, etc.) must be established to maintain cell viability and function according to cell bank requirements. If hematopoietic progenitors are the stem cell source (e.g. bone marrow or peripheral blood), coagulation must be avoided, for example by collecting the cells in acid–citrate–dextrose (ACD) or citrate–phosphate–dextrose (CPD) solutions. Umbilical cord blood is usually collected in a bag containing approximately 23 ml of anticoagulant solution CPD-A and maintained at 4°C until processing. For tissue fragments, a basal nutrient medium [e.g. Dulbecco’s modified Eagle’s medium (DMEM) or Medium 199] can be used as the transport solution, with the addition of antibiotics to avoid the growth of microorganisms. If processing of the tissue fragment is to be delayed, then donor serum (5–10%) can be added to the medium.
In addition to cells or tissues, associated biological samples must be included in the package for the bank in a suitable condition for the intended use, and documentation (informed consent, detailed description of products, data related to the collection process, etc.) must be included and must be legible. External labeling must include the center of origin and person responsible, advice on handling, center of destination and person responsible, etc.
Processing
Upon arrival, the package contents (cells, tissue fragments, samples, documentation, etc.) are checked to assess their suitability. The origin of every individual product must be identified unequivocally to ensure traceability.
The areas where the cells are exposed to the environment during processing are considered critical because of the high risk of contamination, so they must be exhaustively controlled. The specifications on the air quality in the different areas of the bank must be defined and an environmental monitoring system used, according to this classification, which includes a particulate count ( Table 26.2 ) and microbiological cultures ( Table 26.3 ) .
| Grade | At rest | In operation | ||
|---|---|---|---|---|
| Maximum permitted number of particles/m 3 equal to or above | ||||
| 0.5 μm | 5 μm | 0.5 μm | 5 μm | |
| A | 3,500 | 0 | 3,500 | 0 |
| B | 3,500 | 0 | 3,500 | 2,000 |
| C | 350,000 | 2,000 | 350,000 | 20,000 |
| D | 3,500,000 | 20,000 | Not defined | Not defined |
| Grade | Air sample | Settle plates (diam. 90 mm) | Contact plates (diam. 55 mm) | Glove print, 5 fingers |
|---|---|---|---|---|
| (cfu/m 3 ) | (cfu/4 h) | (cfu/plate) | (cfu/glove) | |
| A | < 1 | < 1 | < 1 | < 1 |
| B | 10 | 5 | 5 | 5 |
| C | 100 | 50 | 25 | – |
| D | 200 | 100 | 50 | – |
To maintain the aseptic chain, it is important to use flow cabinets with grade A air quality and at least a grade C background. Depending on the air flow direction two types of cabinet can be distinguished: horizontal, where the air flow blows from the side facing the operator, parallel to the work surface, and is not recirculated ( Fig. 26.1 ); and vertical, where the air blows down from the top on to the work surface and is drawn through the work surface and either recirculated or vented ( Fig. 26.1 ). The first type permits more freedom of movement and the second, which includes a front panel, offers more protection for products and technicians.
To achieve the highest efficiency from stored products, it is very important to prepare them according to optimal processing and storage protocols.
Cell Suspension
Organs and tissues contain different cell lineages with different functions and capabilities. Whether stem cells are present in all tissues is still a matter of debate. For stem cell banking, the cells intended for therapeutic purposes must be selectively isolated and concentrated.
Cells can be isolated from tissue fragments by two techniques. In the first, explant culture, tissue is chopped finely and the pieces are seeded on to the culture surface. It is recommended that a high concentration (30–50%) of serum is used in the nutrient medium, so that surface tension holds the small pieces in place until adhesion. Then, after a few days, outgrowth of cells is achieved by migration from the tissue to the culture surface ( Fig. 26.2A ). Later, when the culture reaches subconfluence, cells can be detached and suspended in isotonic solution. The main advantage of this technique is that tissue is minimally manipulated, but cell yields are lower than those obtained with enzymatic digestion, because some cells in the tissue fragment do not contact the culture surface.
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