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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Regulatory Issues: Down to the Bare Bones

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In this chapter we propose a brief description of the relevant issues that tissue-engineering researchers must consider when planning new therapeutic approaches, especially in the bone and cartilage repair field. An overview of the current regulatory authorities is presented to evidence their role as responsible rule-makers. The influence of the market and of the private enterprises, as well as that of the academic world, is also taken into consideration when defining rules to follow.

We will discuss existing guidelines and legislation, giving an overview of the requirements to be considered when projecting a possible product application in the field of tissue engineering. The reader's attention will be primarily focused on the cell-based composites, being these products the most challenging in terms of application of this approach to bone repair. A general outlook of the requisites for the manufacturing of tissue-engineered products will be discussed, spanning from the origin of the cellular component of the composite to the release criteria of the final product.

The nature of the products or of the therapeutic strategies will not be dealt with, unless they represent useful examples to evidence the related regulatory issues.

Introduction

Progress in cell biology and biotechnology and improvements in therapeutic treatments have made available to clinicians tissues to be used in reconstructive surgery for the repair of extensive lesions. In particular, a role for several growth factors controlling cell proliferation and differentiation has been established and cell culture techniques have been improved to allow the in vitro selective expansion of restricted cell populations. Tissue engineering, i.e., the association of ex vivo grown cells and/or biologically active molecules with different materials, is furthermore driving the production, the testing and the marketing of new generations of transplantable biomaterials. These can now be used in unlimited quantities. Ideally the new approaches based on cell-biomaterial associated products will substitute the previously used devices which do not represent sufficient or satisfactory solutions. Repair of soft tissues - previously hardly feasible - may now be envisioned and performed by transplanting the engineered tissue obtained from the expansion of cells of the patient in association with resorbable materials (either synthetic or of natural origin).

The first “manipulated” cells intended for structural repair or reconstruction were autologous keratinocytes expanded ex vivo for treatment of burn wounds and leg ulcers. Additional examples were chondrocytes cultured and implanted in focal cartilage defects, allogeneic dermal fibroblasts for dermal wound healing and the transplant of specific endocrine cells. Other applications of the tissue engineering approaches presently at a preclinical or very early clinical stage are the repair of skeletal and cardiac muscle fibers, the implant of fat cells for cosmetic augmentation, the realization of vascular prosthesis and the selection of cells with a neuroregenerative potential. Each of these applications may also take advantage of a possible genetic modification of the patient's or of the donor's cells by the insertion of a modified, or repaired or new gene.

Commercial distribution of such products could provide a relatively simple solution to a common type of injuries, satisfying the needs of a large number of patients. When commercial establishments began to provide these manipulated cells to surgeons within the United States it became clear that such a class of products had not been explicitly considered by the FDA. Moreover the manipulation required was much more than that needed for autologous bone marrow transplant, where the source tissue is harvested but hardly “processed” at all.

Tissue Engineering of Bone

Particularly, in the repair of bone and cartilage, the tissue engineering approaches are seeking to address the needs by the realization of viable substitutes that restore and maintain the function of the two types of tissues. With respect to standard drug therapy or permanent implants, the tissue-engineered bone becomes integrated within the patient, affording a potentially permanent and specific cure of the disease state. Any approach involves one or more of the following key ingredients: harvested cells, recombinant signaling molecules and three-dimensional matrices. Ideally the cells would attach to the scaffold and proliferate and differentiate under the control of endogenous and exogenous growth factors to ultimately organize into normal healthy bone or cartilage as the scaffold matrix degrades.

Orthopedic surgeons had previously used autologous tissues (for ex. rib cartilage) without regulatory board oversight. It was immediately obvious that different rules should apply to tissue engineered products. For example, chondrocytes dissociation from healthy tissue and their ex vivo expansion (to reach the proper number for reimplantation) would fall under the somatic cell therapy product definition. Nonetheless, since chondrocytes were implanted in defined lesion sites, contrary to systemic therapies, this treatment showed similarities with some device products.

In defining rules, one must take into the account the followings: i) the need of unconventional culturing techniques. Cells do not organize into tissue or organs simply by culturing them in standard media, nor are they able to acquire the proper phenotype without specific signaling/differentiating factors; ii) the need of appropriate matrices and/or biomaterials that provide the required structural scaffold properties at the lesion site and that allow to be remodeled while the new tissue is being formed. Combination products, derived from somatic cell and/or gene therapy products grown on different scaffolds may evidence different biological properties depending on the scaffold itself; iii) accessibility of the lesion site by the specific surgical techniques needed to implant the cell/biomaterial construct.

Since the chemical/physical properties of many synthetic matrices can be easily defined and controlled and the surgical procedures needed for the new therapeutic applications can be easily overviewed, the real new regulatory challenge is posed by the combination products and the absence of rules for the manufacturing of their cellular components.

With respect to their mode of action, combination products can be divided into two main categories: the metabolic support systems and the tissue repair/replacement products. Products for bone and cartilage reconstructive therapies normally fall under the latter category. Cells are either autologous or allogeneic multi or pluripotent cells—stem cells included—all of which could also be genetically modified. The device part is generally identified as “natural biomaterials” (collagen sponges, amniotic membranes, demineralized bone) or as “synthetic polymers” (PLGA-derivatives, ceramics). They can be used either for temporary or permanent implants in a wide range of applications.

Tissue-Engineered Products: Drugs, Biologics or Devices?

Cell and gene therapies represent new approaches not feasible only a few years ago. They are focused on the patient, in many cases the human subject being the source of his/her own means of cure (autologous cell therapies). The first worldwide authority organization to recognize the need of appropriate guidelines for this type of therapies was the American Food and Drug Administration. It is of relevance that the US attention to the issue came as a direct consequence of the vital framework that interconnects the industry and the academic world in the North American scientific community. Presently several national and international public offices deputed to drug regulation, among which the Center for Biologics Evaluation and Research (CBER) of the american Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products (EMEA), are developing rules and guideline for the manufacturing and use of tissue engineering products.

Initial regulations in the field were derived from the interim rule for banked human tissue promulgated in the American federal register on December 14th, 1993, entitled “ Human tissue intended for transplantation”.1 In this document banked human tissue products are defined as “...derived from a human body which: 1) is intended for administration to another human for the diagnosis, cure, mitigation, treatment or prevention of any condition or disease; 2) is recovered, processed, stored, or distributed by methods not intended to change tissue function or characteristics; 3) is not currently regulated as a human drug, biological product, or medical device; 4) excludes kidney, liver, heart, lungs, pancreas, or any other vascularized human organs; 5) excludes semen or other reproductive human tissues, human milk and bone marrow...”. In addition, recognizing that the sponsors of the developing tissue and cell based therapies were willing to render their products commercially available, another note was issued on the federal register within the same year, defining the somatic cell therapy products as “...autologous (self ), allogenic (intra-species), or xenogenic (inter-species) cells that have been propagated expanded, selected, pharmacologically treated, or otherwise altered in biological characteristics ex vivo to be administered to humans and applicable to the prevention, treatment, cure, diagnosis, or mitigation of diseases or injuries...”.2 In this document a relevant statement was promulgated by the FDA, and widely accepted by the international scientific community: the definition of “manipulation of cells” was described as the ex vivo propagation, expansion, selection, or pharmacological treatment of cells or other alteration of their biological characteristics.2 Clear emphasis was posed on the need to precisely frame a “biological characteristic” (unique and identified by any specific technical means) and its “normal” or “altered” condition.

Nonetheless these regulations were insufficient for a higher scale exploitation of the technology and for its transfer to the market, mainly for the temporary absence of reference standards, of product quality assessments and of lot release controls. Instead, the required screening of the starting material was already partially regulated, taking advantage of the issues dedicated to the transplantation of human tissues.1

Thus the industrial and the private investments world were looked upon as proponent counterparts by the regulatory boards. On the contrary the academic world hardly interfaces with the legislator, traditionally focusing on basic research as a primary goal. Academic scientists may often be applying an innovative approach that clinically works but that was never screened nor approved by any official board and/or national commission.3 It should be stressed that, as a consequence, these therapeutic approaches have grown to the status of “possible marketable products” either from only partially controlled clinical trials or directly off the bench of academic institutions. In this respect, therapies that take advantage of autologous cell culturing often derive the culture conditions directly from the optimizations run on the laboratory benches. Usually several media are tested, with different self-made modifications, indeed needed for specific cell types or for particular growth conditions. Nonetheless, ancillary products, such as growth factors, cytokines or chemicals in the culture media, not intended to be part of the final product, might affect its quality, effectiveness, or safety.

Unfortunately innovative therapies were already shown to carry potential unwanted effects, besides being sometimes unclear regarding the benefits gained by the patients as already tragically experienced in the case of gene therapy.4-7 Furthermore, few preclinical data have been published and toxicological evaluations are seldom found in the literature for the ancillary substances that may still contaminate the final product. Nontissue components, even though to a lesser extent, could also represent means of unwanted effects. It should be stressed that tissue-based products that are intended for therapeutic use, that contain synthetic or mechanical components and that achieve their primary mode of action by means other than metabolic or systemic action are regulated as devices, including combination products. In the United States, their evaluation should therefore be carried out by the Center for Device and Radiological Health (CDRH) of the FDA.8 Premarket investigations and clinical trials would be required under investigational new drugs (IND) or investigation new device exemptions (IDE), as appropriate.

Composite products for tissue-engineering of skeletal tissues are comprised of two or more regulated components, i.e., drug/device, biologic/device, drug/biologic or drug/device/biologic that are physically, chemically or otherwise combined or mixed and produced as a single entity.9 Composites such as a biomaterial associated to bioactive molecules (growth factors, cytokines, etc.) may ultimately be considered as devices exerting their action as a drug, and therefore be regulated by the Center for Drug Evaluation and Research (CDER) of the FDA. Others composites, such as a porous ceramic associated to autologous osteogenic cells, would obviously fall under two categories. Wisely the FDA has recognized the possible overlaps of competence in the evaluation process of these new therapeutic agents. In the same document, therefore, a clear statement was made to sustain the setting of a Tissue Reference Group, composed of six members, three of the CBER and three of the CDRH, to assist in making jurisdictional decisions and applying consistent policy to these products.8 The bottom line is that the mode of action of the combination product, once defined, dictates which FDA branch will review applications and draw the rules for the therapeutic use.

In the “Proposed approach to regulation of cellular and tissue-based products” issued by the FDA it is also stated that “...for combination products with synthetic or mechanical components (which comprise the largest class of combination products), clinical trials and marketing applications must address the clinical safety and effectivness of the overall product, as well as the function and compatibility of the synthetic or mechanical components. The agency principal concerns are that they function correctly, that they last a predictable and adequate length of time and that they are compatible with surrounding tissues...”.8

Thus the real challenge for national and international regulatory boards is how to step into a guideline and law proposal strategy that grants efficiency and at the same time sufficient flexibility to meet the patients' needs and rights, the scientific innovations and the market-oriented enterprises.

The Backbone of the Current Regulatory Frameworks for Cell Therapy Products

This section is a summary of most recent and updated guideline for design/manufacturing of a tissue engineered product to be used for cartilage/bone repair. It should be noted that any nation may have considered or may be considering to introduce, apply and enforce additional regulatory requirements.

In this overview, we will not encompass the current issues that rule the procedures used to manipulate whole organs or minimally-manipulated bone marrow (both of which are under the Health Resources and Service Administration in the US), or the transfusable blood products (i.e., whole blood, red blood cells, platelets and plasma) which are ruled separately. We will not deal also with the current framework for tissue-related products, such as those derived from animals, with products used in the propagation of cells or tissues, or products that are secreted from cells or tissues, (such as human milk, collagen or growth factors).

Cell Therapy Products

In the USA, the Center for Drug Evaluation and Research (CDER) has been assigned as the FDA component that has the primary jurisdiction for the regulation and the premarket approval of the products derived from autologous cells manipulated ex vivo (MAS cells). Clinical investigations of MAS cell products should be performed in accordance with the requirements for investigation of new drugs and they are subject to licensure as biological products. The current Good Manufacturing Practice regulations (GMPs) will apply, although FDA intends to consider on a case-to case basis alternative approaches to specific regulations, when these may be impractical or unnecessary to assure safety, purity and efficacy of the product.10

Establishments engaged in the manufacture of the MAS cell products should register as a drug manufacturer, and should be subjected to inspections on a biannual basis by a representative of the CBER and by an investigator from the closest FDA District Office.

European or Asian nations will refer to the European Agency for the Evaluation of Medicinal Products (EMEA) or to national authorities for regulatory jurisdiction and similar controlling actions. It should be stressed that US officials will not allow marketing within the USA of MAS cell products manufactured in a plant outside USA, unless directly approved by the FDA or unless evaluated through a FDA-approved review process.

Whether the MAS cell product will be applied by public institutions or sponsored by private companies, investigative clinical studies will be required. The FDA guidance on “Applications for products comprised of living autologous cells” also state that “...Prospective randomized clinical studies traditionally have been the best way to demonstrate safety and efficacy. However, where studies of MAS cells without internal patient controls provide evidence of effective structural repair which substantially and clearly represents improvements in outcomes compared to patients in an appropriate historical databases, this may be sufficient to demonstrate efficacy...”.10

To follow the proper guidance for clinical trials, early contacts of the proponents with the relevant authorities are strongly recommended. Previous to any activity, the structures where the tissue engineered products will be manufactured and clinical experimentations will be carried out need to be credited and certified by the competent authorities.11-12 Within the term “structure” several items are included, such as room and building requirements, hardware requirements, as well as legal and clinical/scientific responsible officers.

In several European countries, Ethical Committees within hospitals have the power to approve or refuse clinical trial proposals on the basis of the risk assessment for the patient. They also are the proper administrative body to overview adherence to international and national law requirements including the existence of adequate manufacturing structures. If the use of a new drug/device/biologic is suggested and the risk evaluation is undetermined or unclear, as in most cases of tissue engineered products, the appointed committee may directly request the Ministry of Health and/or national health institutions approval. In the case of Italy, for example, this is the proper role of the Istituto Superiore di Sanita'.13 The Istituto Superiore di Sanita' would require, in turn, immediate interaction with the proponents to establish a controlled phase I clinical study, according to all the GMPs, law and guidance recommendations.

Manufacturing Facilities

Following are some specific points to be considered before proposing a clinical study to prove safety and efficacy of a tissue engineered product.

General Room/Building and Hardware Requirements

  • all cultures for tissue engineering purposes will have to be performed aseptically and by using sterile instruments;
  • source tissue harvesting and cell culturing must be performed in GMP approved rooms. Whenever necessary, as for gene therapy applications or as decided on a case-to-case basis by the appropriate authority, in biosafety containment level 3 rooms (BL3; the rooms must be provided of controlled access, air locks, biosafety cabinets where no work is allowed on open benches; all surfaces must be sealed, all penetrations — telephone, lights, gas, vacuum, electrical line, water - are to be caulked, collared or sealed to prevent leaks, and negative airflow pressure, doublebacked ventilation system and absolute filtration units must be available, working and properly maintained);
  • rooms where cell cultures are performed for clinical use must be used for that purpose only; in the case of research institutions performing a few tissue engineering cultures in a limited period of time, dedication of the same cell culturing rooms to other activities can be accepted by the competent authority upon validation of the cleaning and sterilization procedures prior to cell culturing for clinical applications;
  • rooms must be scheduled for routine cleaning procedures and sterilization; additionally BL3 rooms must have independent accesses; operational procedures in the cell culture rooms must avoid cross contaminations between cells derived from different donors/patients (spatial of temporal separation are strongly suggested);
  • all the hardware used in cell culturing for clinical applications should be provided of specific sensors and visual/acoustic malfunction alarms (i.e., freezers, incubators, laminar flow hoods, cryogenic hardware); backup apparatuses must be available, working and properly maintained);
  • certified procedures must ensure sterility of the outgoing products, either from viruses, mycoplasma or bacteria;
  • certified procedures must ensure the tracking down of any used material in any produced lot of final products;
  • for each manufacturing location a floor diagram should be included that indicates facilities layout. The diagram does not need to be a detailed engineering blueprint, but rather a simple schematic that depicts the relationships between the different rooms of the manufacturing areas and that indicates the use of adjacent ones. The diagram must report the flow of production of the biological substance and any other specific activity in dedicated rooms (for example storage of raw materials, quarantine, etc.)
  • in the absence of any specific regulation, Good Laboratory Practices (GLPs), GMPs and ISO 9002 certifications must be considered as the normal working standard.

For clear economical and technical reasons, controlled areas and rooms are normally included and surrounded by lower-requirement areas (i.e., where the control parameters, such as air purity, sterility, safety containment are less strict). This disposition reduces costs to maintain elevated safety levels. The interfaces between areas of different levels are critical: while planning the production process, fluxes of materials/operations from the interior to the exterior of the different areas must be considered. Segregation of the different areas can be obtained by dislocation (low pressure, high flux) or differential (high pressure, low flux) air pressure between the areas. This concept applies to both sterile and nonsterile productions and is summarized in Figure 1.

Figure 1. Schematic of the fluxes of materials/operations/personnel through controlled contamination areas.

Figure 1

Schematic of the fluxes of materials/operations/personnel through controlled contamination areas. Continuous lines represent means of physical separation (exterior walls, interior walls, transfer compartments) or of contamination containment (laminar (more...)

Personnel Requirements

  • a legal representative, distinct from the scientific/clinic responsible officer, must be identified in each structure;
  • the scientific/clinic responsible officer must have a proven expertise in the biomedical field of interest;
  • a quality control responsible officer must also be identified, distinct from the scientific officer. Only in the case the structure performs a few cell cultures in a limited amount of time the scientific/clinic responsible officer can serve also as quality control responsible;
  • personnel must be aware of the standard working operational procedures as well as of the safety and emergency procedures; in this respect the European Countries are also committed to follow international guidance on the safety issue, as indicated by the european directives on safety measures.14

Product and/or Process Quality Assessment

In a final rule issued in 199615 the FDA has amended the definition of “manufacturer” to include an applicant for a license. The definition broadly encompasses academic laboratories and clinics which would assume responsibility for the safety, purity and potency of the final tissue engineering products even if not directly involved in significant manufacturing steps. By converse, “manufacturing” becomes broadened to include the more-than-minimal manipulations occurring in autologous cell culturing, as already described.

Whether it comes from the academic or the private enterprise world, the proper documentation must be submitted to the relevant authorities for the peer review and approval procedure. So far all official authorities of countries where a tissue engineering activity is being performed have substantially agreed to request similar detailed information for the evaluation process of those products that include cells, either for autologous or allogeneic use. In particular they all request:

  • scientific rationale and objectives of the clinical experimentation;
  • characterization of the starting cell population and of the auxiliary components;
  • procedures used to manipulate the cells and the auxiliary components;
  • description of the cell banks (where needed);
  • characterization of the final cell and/or tissue engineered product;
  • procedures for quality control of lots of the final products;
  • preclinical documentation on the toxicity of the final product;
  • preclinical documentation of the final product efficacy.

To solve the lack of standards, new techniques must be set on a case-to-case basis. Typically the active principle in the cell therapy based products is poorely durable. The available cells are usually scarce and their number not sufficient for extensive quality control tests. In the case of severely aggressive diseases, the use of autologous cells imposes defined temporal limitations: a prolonged expectance before the implant may hinder the patient's condition or alter his/her morbidity to the treatment. In addition cell products normally need to be used few hours to a few days from the time of the product preparation.

Since autologous cells are to be used in the same donor/patient, and given the above mentioned limitations in lot quantities and timing, the number of applicable tests can be reduced. For example the official sterility test of the European Pharmacopea takes no less than fourteen days to be performed. Clearly, the short half-life of cell therapy products may require their use in advance to the availability of test results; nonetheless“...the manufacturer must still employ appropriate controls to provide assurance of safety, purity and potency of MAS cell products...”.10

The requested documentation is necessary not only for the donor/patient's risk assessment, but also for the analysis of the therapy efficacy. For the FDA, in order to use and market a MAS product, there is no need to demonstrate its superiority with respect to other existing drugs/ therapies.10 On the contrary, the Committee for Proprietary Medicinal Products of the EMEA (CPMP), and therefore the national regulatory boards of the European Community countries, require a clear evidence of its effectiveness. In fact the European Committee follows a more restrictive rule, probably considering a higher intrinsic risk as associated to the autologous cell therapy with respect to other approaches.

Risk Assessment Analysis

A proper application and risk management/assessment analysis should take into account all the following factors:8-12,16-17

Origin of Materials To Be Used in the Composite Product Manufacturing

All culturing media, either for cells or tissue cultures, should be certified for their composition, purity, salt and organic component content and indicate a defined expiry date. Similarly all the auxiliary materials which will be part of the final product (i.e., scaffold matrices, biopolymers, ceramic components) or that are used in the manufacturing processes (i.e., plasticware, disposable materials, common reagents) should be certified by their respective producers through identified quality assessments. Lot numbers must be registered. For each chemical used, in addition to the lot number, one must register manufacturer, production date, purchase date, expiry date, composition, purity grade, biological activity and known contaminants.

All material used in the manipulations of the cellular component of the composite should be certified for clinical usage. Unfortunately for many of the culturing media components (cytokines, growth factors, antibodies, etc.) this standard is not available; however, for the ancillary products whose function is necessary only during the manufacturing procedures, a proper assay that determines their absence in the final product would be sufficient to grant their use.

All materials must be checked in terms of sterility, apirogenicity and absence of contaminating agents.

Use of any substance that may induce immune response (animal serum, serum proteins, antibiotics, antimicotic agents) should be avoided or limited to the very first manipulation procedures; indeed these substances must be absent in the final product.

Specific attention should be posed to the use of fetal bovine serum, due to the serum-associated risk of transmission of unknown pathogens. Ultimately the use of human serum, if necessary, must be properly justified.

Donor/Recipient Selection

Donors can be living or deceased. Obviously, for composites that include autologous cells, only living donors are considered. Donor files should include the following:

  • identity code for each donor;
  • name, age and gender; age should be limited, according to the most recent data available, to ensure proper cell response;
  • health status;
  • date and time of the biopsy;
  • biopsy characteristics (procedures applied, size, purpose, conservation means). Logistic of biopsy shipment (sending and receiving institutions, way of travel, etc) should also be recorded.

Donor/Recipient Exclusion Criteria

Donor's cells must be preventively addressed as for autologous or allogeneic use. Allogeneic therapies are associated to a high potential risk of transmission of pathogens. Donors should be excluded if:

  • affected with extended infections or septicemia;
  • positive to syphilis, type B hepatitis, type C hepatitis, AIDS (or if included in AIDS-risk associated categories), HIV-I and/or HIV-II, Creutzfeldt-Jacob syndrome (CJD);
  • affected with neurological diseases of viral or unknown origin;
  • previously treated with the human growth hormone and/or drugs derived from the human hypophysis;
  • previously treated with dura mater;
  • previously treated with demineralized bone; demineralized bone falls under a different regulatory panel, as described in a following paragraph;
  • previously treated with bovine pericardium;
  • affected with malignant tumor(s);
  • affected with genetic diseases that may compromise the recipient tissue response to the composite application;

All the donors/patients will repeat testing 6 months after inclusion in the protocols for those pathologies for which a positive test response is not immediately detectable after infection. For a second screening, alternative tests may also be applied, such as the polymerase chain reaction (PCR), if of proven validity.

Cells or tissues of donors found positive to exclusion criteria should not be acquired. If acquired they should be destroyed. This rule may not apply to autologous transplantation procedures or in rare cases of allogeneic therapies for which either the syndrome gravity or the donor's genotype rarity make other therapeutic approaches unfeasible. In theses cases the competent authority must also certify the conservation procedures of biological material explicitely identified as “biohazardous”; all the procedures for the collection, disposal and conservation of potentially biohazardous material must be described in detail prior to approval.

Prerequisites for Cell Banks (If the Cell/Biological Component of a Composite Is Retrieved from or Storaged into)

The current guideline suggests to preserve any specific and characterized cell population in a cell bank to be able to recover the cell population used for somatic therapies. This may not be feasible for therapeutical applications, such as in the case of cartilage reconstruction where the autologous chondrocytes are few and can be expanded for a limited amount of cycles.18 The “bank” itself should be composed of the “main storage cell bank” (MCB) and of a “working cell bank” (WCB).11 The MCB is defined as the collection of aliquots of homogeneous cell populations that have been previously characterized in terms of genotype, phenotype, biological functions and purity and that, when feasable, were obtained by clonal expansion. The MCB must be preserved in at least three different locations in appropriate liquid nitrogen containers.

The WCB is the collection of the aliquots of an homogeneous cell population obtained from a single aliquot of the MCB via in vitro amplification for a defined amount of time and/or cell cycles. Cell culturing conditions must be described in detail. Aliquots, obtained either from the MCB or the WBC cannot be reinserted in the bank once used. The cellular component of the composite originates from one WCB aliquot.

Procedural operations performed to constitute an MCB or a WCB must be described in detail. Issues related to the cryopreservation of the cells are critical. The specific protocols must indicate:

  • the composition of the preserving solution. Several solutions are commercially available. It is mandatory that cryopreservants are eliminated from the cell suspension before use. A low toxicity at the concentrations used and a total absence of mutagenicity effects are also mandatory;
  • the precise temperature at which cells must be preserved; alarmed and backed-up systems are required for -80°C freezers and liquid nitrogen containers to prevent power and/or temperature failures. Upper and lower limits for working temperatures must be determined;
  • the maximal duration of the preservation time for every manufactured product (to be estimated with proper experiments if not available);
  • operational procedures and timings for freezing and thawing passages (in most cases, cytotoxicity of the cryopreservants at standard room temperature imposes an extremely rapid thawing procedure);
  • the detailed description of all the parameters to assess the cell viability in the in the expanded cell population on a short and long term (colony forming efficiency and life span, respectively). Procedure must grant a defined percentage of viable cells;
  • the precise location of each container. Aliquot amount and identity of each cell/tissue component should be recorded on a specific register.

A proper risk assessment/management should anyhow follow the general scheme proposed in Figure 2 (adapted from EN-ISO 14971).17

Figure 2. Schematic summarizing the risk management/assessment for a GMP/GLP and/or certified procedure.

Figure 2

Schematic summarizing the risk management/assessment for a GMP/GLP and/or certified procedure.

Procedure (Checking in Vitro Expansion and/or Engineering of the Cell Components)

Cell components are potentially subjected to several biological risks during the expansion and/or storage phases; to ensure the manufacturing quality, intermediate and final products should be tested for the following:

Cell Identity

The identity and composition of the cell population through the different manufacturing phases must be determined. A set of genotypical, phenotypical and functional markers must be available to follow and unequivocally identify the proper cell type.18

Contaminating Agents

Risk of bacterial, viral, micoplasmic, fungal or yeast contamination during the manufacturing process and in the intermediate/final products must be reasonably excluded. It is responsibility of the manufacturer to grant the purity and safety of the final product.

In the U.S.A. the FDA requires testing of blood samples from allogeneic donors of hematopoietic stem cells in order to prevent the transmission of communicable diseases. The implementation of regulatory procedures, paragraph A-2, states: “...For peripheral blood stem cell donors, the donor's blood, and for umbilical cord blood donors, the mother's blood, would be required to be tested for HIV, cytomegalovirus, HTLV, syphilis and hepatitis infection....physical examination of perspective donors would include screening for high risk for HIV, hepatitis, CJD and tuberculosis”.8 The same paragraph also states that “..the agency intends to recommend, but not require, that testing be performed when stem cells will be used in the person from whom they were obtained. In such a case the agency would recommend only the following tests: HBsAg, anti-HCV, anti-HIV-1, anti-HIV-2, HIV-1-Ag and HTLV-I/II. The agency also would recommend that the history and physical examination of the donor include screening for high risk HIV and hepatitis. The agency would require that record-keeping and labeling reveal which of the recommended tests were performed and their results, as well as which of the recommended tests were not performed”.

Nonetheless, for tissue engineering purposes, autologous cell preparations can also be obtained from donors resulting positive to some of the exclusion criteria. As for the circumstances justifying the use and storage of such cells or tissues, the agency requires “ 1) that the cells be labeled as “biohazard”, 2) that autologous products also be labeled as “autologous use only”; 3) written advanced informed consent of the recipient be documented; 4) there be documented concurrence of the recipient's physician before the cells could be released from the cell bank.”8

Aging

Primary human cell divide for a limited number of cycles, undergoing a senescence degenerative phase thereafter. A senescent cell may also have lost the biological properties of the young differentiated phenotype that physiologically constitutes the tissue of origin. Senescent cells can be useless for tissue engineering purposes. It is therefore necessary to record number of doublings of any cell population used for tissue engineering purpose, starting with the biopsy and including procedural steps before and after the cryopreservation passage, if performed. The number of the in vitro doubling must not hinder the proliferation capacity of the expanded cells.

Chromosomal Alterations

Prolonged culturing of mammalian cells increases the risk of quali-quantitative chromosomal alterations in the cell genome. Standard karyotyping techniques should be applied to evaluate the stability of the expanded cell population karyotype.

Differentiated Phenotype Loss

During the in vitro culturing cell may loose, partially or totally, the expression of specific markers of their differentiated state. By converse they may acquire the expression of new markers that are not typical of their physiological phenotype. Both of these events may compromise the cells future reinsertion in the tissue of origin. Such an eventuality must be verified by means of immunohystochemical analysis, possibly targeting specific gene products of the tissue under investigation (for example keratins for the epithelia). The possible existence of a contaminant cell population harvested at the time of the biopsy—able to propagate and progressively substitute the cell population of interest—must also be exploited. Immunocytochemistry and PCR techniques are the suggested approach to tag and check this eventuality.18

Neoplastic Phenotype

The neoplastic transformation is a rare phenomenon in mammalian and human cells. Nonetheless the exposure to several ancillary products (cytokines, hormones, growth factors, viral vectors) may alter the cell predisposition and increase the risk of neoplastic occurrences. Absence of risk must be assessed before the use of the cell/composite product in humans either by in vitro culturing techniques or by transplantation in immunodeficient animals. Assays should be planned for the process quality assessment if not feasible for each different human autologous culture.

Quality Controls for the Final Product19

Purity Quantity and Biological Activiy

It is necessary to define the required dose of the manufactured product to obtain the biological/therapeutic effects. Doses (for example the average cell concentration in a given composite product) may be derived from preclinical experiments or may vary according to each donor's cell responsiveness to culturing conditions (typically cell response depends on the donor/patient's age/health condition).

Culture conditions may also influence the cell(s) properties or responsivness to the ex-vivo expansion; it is therefore necessary to assess the quality of the cell product by means of appropriate markers. In the case of cultured chondrocytes, for example, a certified counting method would ensure proper delivery of a correct amount of expanded cells for reimplantation purposes. At the same time, as already mentionned for the control of the cell identity of possible phenotype loss, culturing conditions must ensure the maintenance of a collagen type II producing phenotype in the manufactured chondrocytes.18 The determination of this typical cartilage marker can be assumed as one of the possible standard assessments to be included for the validation of the manufacturing procedures. Clearly it would not be wise to consume the final product (i.e., manufactured chondrocytes) to demonstrate their collagen type II expression, leaving none to reimplant in the donor/patient. Therefore down-scaled tests should be demonstrated as proper and applied when feasible. Alternatively, the quality assessment procedure must be validated. Obviously proper markers must be identified for each specific product.

The biological activity of the final product can also depend upon the site of the therapeutic application with respect to the location of the tissue of origin of the cell component. In this respect the FDA operates a distinction between homologous and nonhomologous function whether or not the product is a structural tissue.8 A structural tissue engineered product is exerting an homologous function when used to replace an analogous tissue that has been damaged: examples include bone allograft obtained from long bones but used in a vertebra; skin allograft from the arm but used on the face and so on.

An example of nonhomologous use is cartilage placed under the sub-mucosa layer of the urinary bladder to change the angle of the uretra and thus preventing the backflow of urine: the cartilage would be acting as a structural support in a district where that support does not normally exist. Another example of nonhomologous use of a cellular product would be the treatment of metabolic disfunctions with stem cells, intended to perform a different function other than the hematopoietic reconstitution. It is evident the need of increased quality controls either for the manufacturing process or for the final products in case of tissue-based products that are used for nonhomologous functions.

Toxicity Studies11,20

Toxicity studies must be performed, as for pharmaceutical drugs. In certain cases the use of human cells in living experimental animals may induce toxic/immuno response due to the inter-specie differences. Therefore proper toxicity studies on the final product must be conducted in immunodeficient animals. Proper species must be selected to give an experimental answer as close as possible to that of humans. For example if the new therapy is based on the effects of a cytokine secreted by transplanted cells, tests should be performed with animal cells that possess receptors for that molecule and where that molecule induces the same biological effects. Undesired toxic effects in body districts other than the ones subjected to the therapy must be clearly reported.

Biological Product Stability

A detailed description should be provided of the storage conditions, along with study protocols and results supporting the stability of the lot/batch of the final product. Stability data must be submitted for the final product as packaged in the container in which it is to be shipped/marketed. If the product is to be shipped under conditions in which cell viability is to be preserved, data must be given to confirm the final product stability under the scheduled conditions. Detailed information must be provided concerning the supplier, address, compatibility results and biological tests.

In the receiving center, an analysis of the container, its closure system, its compatibility with the biological product together with the expiration date should be performed. For sterile products evidence of container and closure integrity is mandatory.

Release Criteria

To evaluate manufacturing consistency and standard adequacy of the products, at least three set of results of quality control tests, performed on independently manufactured complete products, must be submitted under the supervision of the officer responsible for the clinical application.

The documentation should include release criteria, i.e., all procedures and parameters needed to evaluate product transportation condition and its integrity and sterility. In this respect temperature and pH indicators and means of visual inspection of the final product security are recommended. Procedures for release refusal must also be planned.

Demineralized Bone

A separate evaluation is given for the demineralized bone. Decalcified freeze dried bone allografts are considered as unclassified devices, rather than tissues. This view is due to the more-than-minimal manipulations needed to obtain the final manufactured product. The device definition only applies if the allograft is processed to demineralize and preserve the bone to be used as a bone filler in orthopaedic and/or dental applications. In this case, as a Class I medical device, bone allograft would be exempted from premarket notifications and GMP requirements. In contrast, powdered bone (freeze dried bone allografts) falls under a different legislation21 being defined as a minimally-manipulated tissue since the process does not change the integral structure of the tissue.

Conclusions

The emerging field of tissue engineering is rich of activity and promises. In the bone and cartilage repair area many of the tissue-engineered products are based on the use of a cell component associated to a natural or synthetic biomaterial. As for classical pharmaceutical drugs, the criteria for their manufacturing need to be refined and updated in the light of the achievement of the safest and most efficient product. It must be considered, though, that the cell components of the composites are extremely complex open systems,22 on which our present knowledge and control are much more limited than the ones needed to perform a rather simple chemical synthesis. A great deal of research, data and understanding are therefore still needed to improve the state of the art in the field. This involves the public institutions and the private enterprises, but it also calls in for public discussions. New therapeutic approaches pose several ethical questions along with technical doubts, as in germline therapy for example, but they also demand solutions to widespread debates before society comes to their acceptance.23 In this respect the function of the regulatory boards in the different nations becomes extended not only to define the rules, the standards and the manufacturing requirements but also to inform the public opinion and represent it.

In the light of the new role that the patient will have in the future, possibly being the means of his/her own cure, regulatory authorities have to take into account all the legal issues to protect the patient/donor's rights. In spite of the differences of the legal, medical and political issues in the different countries a wider distribution of the same standard techniques and references will make possible for experts to compare different protocols and/or products, allowing a better evaluation of the therapeutic advantages for the patients.

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