Human-derived tissues may be sourced from normal healthy donors,cadaveric donors,or diseased patients,such as those with cancer.Applicable guidelines and standards for the procurement of human tissue are available from the American Association of Tissue Banks (AATB)and the FDA.Additionally,the federal policy in 45CFR Part 46is applicable to all federal or federally supported research.This policy requires that a certified institutional review board review and approve use of any tissue taken from a live human donor.The policy also includes special considerations for research on prisoners,children,and pregnant women or research in other areas involving gestational tissue.In all cases,appropriate written consent must be obtained from the donor or the donor's next of kin,describing which tissue is being procured and for what use it is intended.The donor must meet established guidelines for donor suitability and be tested for the infectious diseases listed in Table 4.The medical history of the donor must be reviewed to ensure the absence of signs and symptoms of these diseases and to rule out issues and behaviors that increase the risk of exposure to such diseases.

Human tissue should be obtained under environmental conditions and controls that provide a high degree of assurance for aseptic recovery.Standard hospital operating room practices are applicable for tissues requiring dissection and surgical procurement.The air quality provided in a typical limited-access operating room is adequate for such procedures.Procurement personnel must be appropriately trained in all aspects of tissue recovery,such as surgical scrubbing,gowning,operating room behavior,anatomy,surgical site preparation,and antisepsis.Special care is required when tissue or organ procurement requires extensive manipulation of the bowel and when sharp dissection may result in the inadvertent puncture of the bowel.Tissue that contains microbial flora (for instance,skin)at the time of procurement can be adequately disinfected by using antimicrobial or bactericidal agents and extensive scrubbing.

Table 4.Infectious Disease Testing for Human Cells and Tissues Used in Cell Therapy Products

Hematopoietic progenitor cells represent one of the most extensively used cell sources in the field of human transplantation.These cells can be collected from the bone marrow,peripheral blood,placental umbilical cord blood,or fetal liver.The source of cells is somewhat dependent upon the patient,the disease,and the clinical protocol.Regardless of the cell source,methods for processing the cells are similar.

Human-derived blood cells and bone marrow cells may be sourced from normal,healthy donors or patients with hematological disorders.Applicable guidelines and standards for the collection and processing of these materials have been published by the American Association of Blood Banks (AABB),the Foundation for the Accreditation of Hematopoietic Cell Therapy,the National Marrow Donor Registry (NMDR),and the FDA.Similar issues regarding consent,infectious disease testing,and donor medical history apply in the sourcing of blood-or bone marrow-derived cells for allogeneic transplants.In cases where these cells will be subjected to selection,expansion,genetic manipulation,or other complex processing procedures,the testing outlined in Table 4should be followed.

Bone marrow for clinical use is harvested predominantly by percutaneous needle aspiration of the anterior or posterior iliac crests or the sternum.Standard hospital operating room practices are employed by specially trained personnel.Plastic syringes and commercially available aspiration needles are used to draw 3-to 5-mLvolumes of marrow from each site of penetration.The material is transferred to a sterile,balanced salt solution or tissue culture medium containing sufficient anticoagulant,such as heparin,to prevent clotting.Removal of bone spicules may be accomplished by passing the material through stainless steel mesh screens or collection kits consisting of sterile,plastic collection bags with in-line filters having about a 200-µm porosity.The volume of marrow collected is dependent upon the body weights and other characteristics of both the donor and the recipient.The maximum volume to be harvested from a donor is about 10to 15mLper kg of body weight.

Circulating hematopoietic,peripheral blood progenitor cells (PBPCs)comprise a small population of peripheral blood mononuclear cells that can be utilized in place of or in addition to bone marrow.PBPCs are collected by apheresis,a procedure by which donor blood is withdrawn from a vein and separated ex vivo into some or all of its component parts.One or more of the components are retained as the harvest and the remaining parts are returned to the donor.Conditioning of the donor may enrich the number of circulating PBPCs in the harvest.Examples of such conditioning include collection during recovery from myelosuppressive chemotherapy and administration of hematopoietic growth factors,such as granulocyte colony-stimulating factor (G-CSF)and granulocyte–macrophage colony-stimulating factor (GM-CSF),or steroids.Collections are also improved by increasing the frequency or volume of apheresis.Apheresis requires one or two large-bore peripheral venous catheters in the upper extremities or a single large-bore,thick-walled,central venous double or triple lumen catheter (Mahurkur type).Two types of apheresis technology are available:the discontinuous-flow cell separators (Haemonetics)and the continuous-flow systems (COBEor Fenwall).Anticoagulation for normal to high flow rates is with a citrate-based material.In a closed system,the risk of contamination is low.The procedure is generally performed by trained,dedicated staff in a blood bank or in a donor center associated with a blood bank.

Placental and umbilical cord blood provides a third source of hematopoietic progenitor cells.Compared to bone marrow and PBPCs,the stem cells of placental and umbilical cord blood have a higher proliferative and self-renewal capacity.Volume of collection and thus cell number are limited and depend upon timing and the presence of a dedicated team of personnel.Collections are made during the third stage of labor.Typically,a closed method of collection is employed and involves cannulation or puncture of the umbilical vein with subsequent collection into plastic syringes or blood collection bags containing citrate-based anticoagulant.The procedure is performed in a controlled-access room away from the site of birth.Cellular content of the collection includes large numbers of erythrocytes,leukocytes,platelets,and target mononuclear cells.An open collection technique,which involves drainage of the blood by gravity from the cut end of the cord into sterile tubes containing anticoagulant,does not afford the same aseptic assurance level as the above-mentioned technique.

Amajor area of concern with the use of placental and umbilical cord blood relates to potential risks of unknown genetic disorders that may be transmitted to the recipient.Donor suitability is established by the usual infectious disease screening of the mother and the completion of a medical questionnaire.The donation remains anonymous and without any long-term follow-up of the child.

The major area of concern with the use of animal tissue relates to the known and unknown risks of potential infectious disease transmission to humans,and as such,the transplantation of animal cells raises unique public health concerns.Introduction of xenogeneic infectious agents into and propagation through the general human population is a risk that must be addressed.Draft Public Health Service(PHS)Guideline on Infectious Disease Issues in Xenotransplantation(August 1996),and any other related regulatory documents that are generated as this field advances,must be consulted when developing xenotransplant cell therapy products.Developers of such products should understand that the product recipients will be subjected to a high level of scrutiny (for instance,clinical and laboratory surveillance or registry in xenotransplantation databases)because of the above-mentioned public health concerns.

The use of animal tissue in the manufacture of cell therapy products requires that the tissue be sourced in a controlled and documented manner and from animals bred and raised in captivity in countries or geographic regions that have appropriate national health status,disease prevention,and control systems.In addition,the care and use of animals should be approved by a certified institutional animal care and use committee.Donor animals must have documented lineage,be obtained from closed herds or colonies,and be under health maintenance and monitoring programs.The facility for housing these animals should be USDAcertified (large vertebrate animals)or Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)certified (small vertebrate animals)and should meet the recommendations stated in the Guide for the Care and Use of Laboratory Animals(National Research Council,1996),which can be obtained from the AAALAC.Such facility should be staffed with veterinarians and other trained personnel who will ensure animal health and disease prevention.The procedures employed in the facility should be documented and records should be kept.Health maintenance and monitoring programs are based on standard veterinary care for the species and include physical examinations,monitoring,laboratory diagnostic tests,and vaccinations.Use of a stepwise batchor all-in–all-outmethod of movement of source animal through the facility,rather than the continuous replacement movement,is recommended.It allows the decontamination of the facility prior to the introduction of the new set of animals,thereby reducing the chance of disease transmission.Feed components should be documented and should exclude,whenever possible,recycled or rendered materials that may have been associated with the transmission of prior-associated diseases.

To provide a high degree of assurance of product safety,screening of donors and of tissues derived from these donors should be performed at several stages throughout the process to rule out the presence of microbial agents.These control tests should utilize assays that are sufficiently sensitive and specific to detect bacteria,mycoplasma,fungi,or viruses of interest.Donor animals can be screened for certain diseases prior to donation of tissue by applying a variety of serological monitoring tests.Tissues can be subjected to a panel of tests including,but not limited to,the following:

Most of these tests are addressed under Analytical Methodologiesor under Biotechnology-Derived Articles á1045ñand Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin á1050ñ.Post-tissue retrieval necropsies,sentinel animal programs,and archival storage of donor organs,tissues,blood,and other specimens are additional components of the overall program to ensure the safety of animal tissue for use in cellular therapeutic applications.

Most of the same aseptic procurement issues apply to animal tissue and to human tissue.Again,the tissue should be obtained under environmental conditions and controls that provide a high degree of assurance of aseptic recovery.Specifically designed procurement facilities,usually closely associated with the animal holding facility,are typically employed.These facilities have specific attributes and design features that may not be available or applicable in the hospital operating-room setting.Such features include the following:(1)staging of various events,such as shaving,sedation,and operating-room preparation,in different rooms that are often separated with air locks for environmental control;(2)high-efficiency particulate air (HEPA)filtration;(3)adjacent but separate facilities for further tissue processing;and (4)dedicated areas for carcass removal.The issues regarding the training of personnel,bowel manipulation and puncture,and disinfection that are applicable to human tissues apply to the surgical procurement of tissue from animals as well (see Human Tissue).

The general principles for processing human and animal tissues following aseptic procurement are independent of the tissue source.The manufacture of cell products may occur at a clinical site or at a central cell-processing facility.Sites involved in cell processing should ensure reproducibility and safety of the manufactured products through appropriate QCand QAprograms.

Regardless of the location,processing should occur in a dedicated area physically separated from the site of procurement.To the greatest extent possible,the facility design and processing procedures should be consistent with those provided by the FDA's Guidelines on Sterile Drug Products Produced by Aseptic Processing(June 1987),or provided under Pharmaceutical Compounding—Sterile Preparations á797ñfor processes involving open manipulation.Generally,this requires that properly trained and outfitted processing staff handle blood or tissue samples in a critical zone supplied with class 100HEPA-filtered air,which is provided by a biological safety cabinet located in a controlled clean room supplied with class 10,000HEPA-filtered air.The facility and processing areas should be monitored for air quality in a manner that provides a high level of process asepsis.For guidance in this area,see Microbiological Evaluation of Clean Rooms and Other Controlled Environments á1116ñ.The material should be packaged in sterile,leak-proof containers and transported from the procurement area to the processing area under controlled conditions that maintain cell viability.The fluid medium in which the specimens are bathed during transportation should be optimized to maintain cell and tissue viability.This transport medium can be supplemented with antibiotics.If so,the antibiotic levels in process buffers are decreased and eventually eliminated during subsequent processing steps,so that antibiotics are not present in the final cellular product.In the case of blood products or tissues containing substantial amounts of blood,the transport media or buffered electrolyte solution should contain an anticoagulant such as heparin or a citrate-based material.

Solid organs or tissues are usually dissected to expose a desired region.This material may be used as is for transplantation or it may be processed further.If multicellular organoids (for instance,islets of Langerhans)or single-cell suspensions are desired,the tissue may be subjected to mechanical or enzymatic disaggregation.Physical disaggregation may be accomplished through the use of instruments that impart high shear forces on the material (namely,to homogenize)or break the tissue into smaller pieces.Alternatively,the material can be pressed or passed through screens of defined mesh sizes.

Enzymatic digestion of the extracellular connective tissue,which holds cells together within the tissue,is another common method for dissociating solid tissue.Typically,the tissue is minced into small cubes,usually larger than 1mm3,and incubated in a buffered solution containing a digestive enzyme.Alternatively,the intact organ is infused with a solution to rinse the blood from the tissue followed by the enzymatic solution that aids the digestion.Various enzymes are used to accomplish this.Examples include collagenase,trypsin,elastase,hyaluronidase,papain,and chymotrypsin.Enzymes with nuclease activity,such as deoxyribonuclease,may be added to digest nucleic acids released from damaged cells,preventing excessive cell clumping.At the end of the incubation process,the cell suspension may be subjected to a mild pumping action to further break up multicellular clusters into those of desired size or composition.Enzymatic and physical disaggregation methods are often combined to achieve the desired result.

Because cells isolated from blood and bone marrow products are inherently cell suspensions,mechanical manipulation is limited to plasma removal,which is accomplished by centrifugation and physical removal of clots that occurred during transport via 200-µm filtration.

Cell suspensions at this stage may be transferred directly to culture vessels as described for Propagationunder Cell Propagation and Differentiation,genetically manipulated as described under Introduction of Genetic Material into Cells,or formulated by various techniques as described under Formulation of Cell Therapy Products.Cell suspensions often consist of a mixture of cell types that may require further processing to isolate a cell population of interest or to decrease the level of an undesirable cell type such as potentially contaminating tumor cells.Various cell isolation and separation techniques exist that provide high yields of pure cell populations.

Each cell type typically possesses specific size and density;therefore,different cell types will sediment at different rates in a centrifugal field or at unit gravity.Cell populations can be selectively sedimented to yield pure fractions by varying the centrifugation forces and the duration of centrifugation.Separation can also be achieved by isopycnic centrifugation,where the cell suspension is centrifuged in a gradient medium that encompasses all of the densities of cells in the sample.In this procedure,the various cell populations sediment to an equilibrium position at the gradient density equal to the density of the cell population.Specifically designed continuous-flow elutriation centrifuges separate cell populations by subjecting a cell suspension to opposite centrifugal and fluid stream forces in a special chamber within the centrifuge rotor mechanism.Cell populations separate within the rotor on the basis of their various sizes and densities,and they are selectively eluted out of the rotor chamber by increasing the fluid stream force.Finally,methods that do not require centrifugation but instead involve the addition of high-density agents,such as hydroxyethyl starch,to the cell suspension will result in cell separation.The mixture is allowed to settle in a tube at unit gravity,resulting in the separation of different cell types based on buoyant density.Concentration and separation procedures such as these frequently result in cell loss due to clumping and aggregation.

Cell separation can also be achieved by applying techniques that take advantage of unique cytological or biochemical characteristics of different cell populations.Soybean agglutinin binds to and agglutinates cells that bear a particular carbohydrate moiety expressed on mature blood cells,but not stem cells,allowing for purification of the stem cells.Lymphocytes possess the CD2antigen that acts as a receptor for sheep red blood cells.The lymphocytes form rosettes,which then can be separated via differential centrifugation.

Some applications take advantage of the ability of certain cell populations to adhere to the surface of specific solid substrates such as tissue culture plastic,collagen-coated materials,and natural and synthetic polymeric scaffolds.The specifically bound cell type is selectively recovered onto the surface and removed from the initial cell suspension.When placed under the appropriate culture conditions,these cells will multiply and eventually occupy the available surface or void volume of the substrate.

Monoclonal antibodies directed against specific cell surface antigens or receptors can be used for both positive and negative cell selection.For example,a monoclonal antibody–labeled cell population can be removed from the cell suspension immunomagnetically,after exposure to magnetic particles coated with antimonoclonal antibody.The magnetic particles and their bound cells are removed from the cell suspension magnetically.Cells are released from the complex following incubation with reagents,such as specific peptides,that dissociate the monoclonal antibody from the cell.Unlabeled cell suspensions can be poured over or incubated on surfaces such as plastic flasks or microspheres coated with monoclonal antibodies as a means of isolating particular cell populations.In addition,a fluorescence-activated cell sorter (FACS)can be used to separate different cell types by binding antibodies tagged with fluorescent markers to a particular cell type.

Various other techniques purify particular cell populations by destroying unwanted cells present in the mixture.For example,certain cell-bound monoclonal antibodies are able to fix and activate complement,which is added to the cell suspension,resulting in lysis of the cell.Some procedures use cytotoxic agents or mitotic inhibitors to selectively impede or kill unwanted cells in a cell product.These methods typically target an unwanted cell subpopulation with a high growth rate,such as tumor cells.Finally,an antibody can be conjugated to a toxic moiety,such as ricin,allowing delivery of the cytotoxic agent to the targeted cell population.Most of these procedures require several washing steps after the exposure of the cells to the cytotoxic agents to ensure the removal of the dead cells,cell fragments,and cytotoxic agents from the final cell product.

Akey issue for cell therapy products is the ability to manufacture and deliver a therapeutically relevant dose of the required cell population to the patient.Depending on the application,the product may be a pure,homogeneous cell type or it may be a mixture of different functional cell types.Many target cell populations are present at low level or low purity in complex primary source tissues.In such cases,production of a therapeutic dose may be achieved only by specific enrichment and propagation of the required cells.

Propagation of cells may occur in suspension culture (for example,Tcells or hematopoietic stem and progenitor cells),adherent culture (for example,mesenchymal stem cells,embryonic stem cells,neuronal stem cells,or dermal fibroblasts),or a mixture of both (for example,bone marrow stroma expansion).Numerous devices of varying degrees of sophistication and automation exist for cell culture.

In the simplest iteration,cells can be propagated in tissue culture flasks (Tflasks),roller bottles,on polymeric scaffolds,or nonrigid,gas-permeable bags inside regular incubator units that are controlled for temperature,humidity,and gas composition.Multilayered plastic cell factories,cell cubes,and multi-bag systems have been developed that enable expansion,harvesting,and formulation to be carried out in a closed system.

Traditional small-scale fermenter units can be used for expansion of cells in suspension culture.It is also possible to expand adherent cells in such units either by providing a surface for attachment (coated beads or disks)or by adapting the cells to propagate in suspension culture.Some culture systems are specifically designed for the propagation of cells for therapeutic applications.These systems tend to be closed systems that use disposable bioreactor cartridges,such as those made of hollow fiber or molded plastic,in automated processing units with direct control of parameters such as temperature,gas composition,and media perfusion rate.These units can provide a completely automated,closed system for expansion and harvesting.In some cases the automated software is set up for patient–donor tracking and will document culture conditions and manipulations for the entire processing run.These features are useful in the design and implementation of QCproduct-release testing programs and for the QAdocumentation of processing runs.

In the case of adherent culture,the cells are usually released from the surface upon which they have expanded.Methods of release include physical agitation,enzymatic cleavage with enzymes such as porcine or bovine trypsin,collagenase,or dextranase,chelation of metal ions (for example,with edetate disodium),and competitive inhibition of adhesion or matrix molecules.As described above,consideration must be given to the source,safety,toxicology,and residual testing for any reagent used to release adherent cells during manufacturing.

Some product-specific systems that do not require the release of adherent cells have been developed.In these systems,the cells are expanded upon a synthetic or natural matrix that is then applied topically (for example,in dermal repair products)or the cells are grown inside or outside of fibers for ex vivo perfusion (for example,hepatocytes in hollow-fiber devices to treat liver disease).In these applications,the matrix and device composition must be biocompatible and,in some cases,biodegradable.

In all of the above systems,standard cell culture parameters must be optimized for maximum process efficiency.Such parameters include composition of cellular source material,initial seeding density,media composition,rate of media exchange,temperature,gas composition,and rate of delivery.Depending on the nature of the product,the potential effect of process parameters on the potency and function of the target cells should be defined.

In closed bioreactor systems,it can be difficult to observe or sample cells so as to determine and control the rate of proliferation and thereby the point of harvest.Measurement of traditional fermentation parameters,such as rate of nutrient usage or production of metabolic products,can provide a surrogate method,amenable to validation,with which to evaluate the rate of proliferation and predict when to harvest the cell product.The relationship of such parameters to the viability,potency,and function of the cell product should be well defined.Postexpansion purification and enrichment of target cells by using methods such as those described above may be required.

Some cell therapies require lineage or functional differentiation of the source cells.For example,hematopoietic stem cell expansion processes normally result in products containing a mixture of multipotent stem cells,lineage-committed progenitor cells,and lineage-differentiated cells.The composition of these products can be manipulated by using different combinations of growth factors and cytokines during the expansion process.The inverse is true for processes in which mature cells are de-differentiatedto enable them to then be recommitted to a lineage pathway (for example,chondrocytes in cartilage repair).

Specific examples of ex vivo manipulation are the programming of professional antigen-presenting cells,such as dendritic cells and monocytes or macrophages,and the production of antigen-specific Tcells to target various specific disease indications.In these applications,the manipulated cells may be engineered to target and attack a specific tumor or tumor cell type,to induce a specific antibody or other cellular response,or to potentially vaccinate a patient.The processes for production of such products can involve one or more exposures of the relevant cells to disease-specific synthetic immunogens (for example,peptides)or natural immunogens (for example,dead tumor cells,viruses,cell membrane fractions,or purified natural molecules)before,after,or during culture expansion.Alternatively,the target cells may be genetically engineered with a specific gene product,such as an HIV-specific receptor.In some applications,relevant cells are cocultured with tumor cells,other diseased cells,or cells producing a transduceable or transfectable gene construct to generate a specifically targeted product.

Again,prior to delivery,the manipulated target cells may require further purification and enrichment by applying the methods described throughout this section.In the case of certain T-cell products,the desired antigen-specific cells can be cloned and then further expanded to provide the therapeutic dose.

Formulations for cell therapy products depend upon the desired length of storage and whether the cells are administered as a suspension or in combination with a matrix.Regardless of the route of administration,cells that will be administered as a suspension can be frozen or not frozen.The most common formulation for cells that are cryopreserved is a 5%to 10%solution of dimethyl sulfoxide (DMSO),with or without hydroxyethyl starch (generally 6%),and a plasma protein,such as 4%to 10%human serum albumin,in a balanced salt solution.DMSOprevents dehydration by altering the increased concentration of nonpenetrating extracellular solutions during ice formation at the time of freezing.The high molecular weight polymeric hydroxyethyl solution protects the cells from dehydration as water is incorporated into the extracellular ice crystals.The use of protein often results in maximum recovery and viability of cells after thawing.Serum (5%to 90%)has been used in place of specific proteins.Some cryopreservation formulations are completely free of protein.If the solution contains a buffer,the pHof the buffer should not be affected by changes in temperature.The optimal concentration of cells for cryopreservation depends on the cell type,but it generally ranges from 106to 107cells per mL.The purity of the cell population can also affect recovery.For instance,granulocytes can be damaged by the cryopreservative and the cell viability can decrease.These effects are dependent upon the concentration of cryopreservative.Both effects subject the patient to an increased level of infusion-related toxicity,although this is related to the volume administered and the final concentration of the cryopreservative.

Formulations for cell suspensions stored without freezing generally contain cell culture media,often without any protein.Because cells continue to metabolize their media even at the reduced temperatures used for storage,the medium supplies the amino acids and other nutrients that help in maintaining cell viability.

Many cell therapy products are administered in combination with a biocompatible matrix.For instance,wound healing or skin substitute products contain cells seeded on a matrix.The biochemical and physical structure of the matrix and the method for combining cells with the matrix are specific to the application.Some common examples include the following:

When manufacturing such products,the primary consideration is the sourcing of a quality matrix material.The matrix material should be biocompatible,should not interfere with cell function,and should not trigger an immune response in the patient.If it is intended that the cells proliferate after loading onto or into the matrix,the matrix and the supporting culture system must allow exchange of nutrients and waste products.Cells may form tissue-like structures under favorable conditions and for those applications where this is required.Athick,impermeable matrix will lead to forming regions of necrotic tissue.Many of these devices are designed so that they can be removed from the patient after a certain period of time.

In all cases where cells are combined with biocompatible matrices,the use of closed systems for the manufacture and the delivery of product is preferable.As cell therapy products of this type can be quite intricate,the manufacturing details for such products are outside the scope of this chapter.