Page 1741 - Hematology_ Basic Principles and Practice ( PDFDrive )

1546 Part IX Cell-Based Therapies

number of supplements have been used during this phase, including reported success growing MSCs in serum-free media or using platelet
CD40 ligand or poly(I:C), interferon-α, tumor necrosis factor-α, lysate, autologous serum, or human AB serum as alternatives to fetal
IL-6, IL-1α, and prostaglandin E 2. Some protocols add proinflam- bovine serum. The traditional method for producing MSCs in tissue
matory mediators at the initiation of the cultures rather than after culture flasks is laborious and involves many open procedures that
induction of differentiation. One issue for manufacturers is finding could result in product contamination. An alternative method is to
clinical or GMP-grade cytokines and growth factors. Some sources use a functionally closed, hollow-fiber bioreactor, such as the
now are available, and many manufacturers assist investigators by Quantum from Terumo. In this system whole marrow is loaded into
providing information on test procedures and stability information. the reactor and the adherent cells attach to the fibers. Nonadherent
Attempts have been made to expand DCs in serum-free medium with cells are flushed from the fibers, which are perfused with the culture
varying degrees of success; improved growth and maturation have medium. Growth is monitored by glucose consumption and/or
generally been obtained in the presence of human AB or autologous lactate production, and the flow rate of medium is increased based
serum. Of note, minor changes in composition of the culture medium on these readings. The cells are removed from the fibers by routine
and even in the type of vessel used for culture have been reported to enzymatic treatment.
affect the yield, phenotype, and functional activity of the resulting Mesenchymal stromal cells can be characterized by their expres-
DCs. Cultured DCs have been effectively transfected using native sion of CD105, CD73, and CD90 and their lack of expression of
tumor DNA or lentiviral or adenoviral vectors. The cells can be CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR.
cryopreserved for later administration or use. They must be plastic adherent under standard culture conditions and
Assessment of DC product usually involves immunophenotyping must be capable of differentiating into osteoblasts, adipocytes, and
+
+
+
(CD1a , CD80 , CD83 ) and some type of functional assay (ability chondroblasts in vitro. Differentiation assays involve growing MSCs
to generate an allogeneic T-cell response or response to a recall antigen under conditions that promote differentiation along the specific
as measured by proliferation assay) in addition to the standard release pathway. For neurogenic differentiation, the medium contains lin-
assays. oleic acid, platelet-derived growth factor, and epidermal growth
factor. Neural cells are identified by immunostaining with antibodies
against tubulin BIII, synaptophysin, galactocerebroside, neurofila-
Mesenchymal Stromal Cells ment M, and neuronal nuclei. To assess adipogenic potential, the
medium contains dexamethasone, isobutylmethylxanthine, and
Mesenchymal stromal cells (MSCs) are cells with multilineage poten- indomethacin. The lipid droplets in the generated adipocytes are
tial that have shown efficacy in promoting engraftment and suppress- visualized by staining with Sudan Black IV. Osteogenic differentia-
ing GVHD. They are also widely used in regenerative medicine tion is measured by culturing the cells in medium containing dexa-
applications, where their mode of action is not well understood. In methasone, β-glycerophosphate, and L-ascorbic acid 2-phosphate.
vitro MSCs are capable of differentiating into bone, cartilage, cardiac Calcium accumulation and alkaline phosphatase activity in the result-
and skeletal muscle, neuronal cells, adipose and connective tissue, and ing cells is visualized by alkaline phosphatase/Von Kossa staining, and
tendons. The cells are not inherently immunogenic and do not appear the osteogenic differentiation is measured as the percentage of min-
to be recognized by allogeneic T cells or NK cells. They express very eralized area in the total cultured area.
low levels of MHC class II and intermediate levels of class I antigens. The ability of MSCs to suppress an MLR is used as an indication
They appear to be able to suppress T-cell proliferation and function of their immunosuppressive activity. A traditional MLR assay is used;
of both memory T and naive T cells in vitro, as well as inhibit the however, in the test wells, the MLR is performed on a layer of MSCs
development of monocyte-derived DCs in vitro. seeded the day before. The response is traditionally measured by
A number of technical variables affect MSC culture and expansion uptake of tritiated thymidine. Numerous animal assays for MSCs are
ex vivo. They include culture medium, passaging density, serum type available but are predominantly used in preclinical studies and are
and concentration, population selection, culture vessel, and use of not useful as release assays. Release criteria consist of the usual assays
growth factors. To reduce some of this variability and decrease the for sterility, endotoxin, and mycoplasma, with immunophenotyping
cost of preclinical testing, the FDA often encourages investigators to for the MSC population. Depending on the intended use of the
adopt a manufacturing process that is in current clinical trials. The MSC, the release process may require an assay showing potential for
following is a basic procedure. MSCs are usually isolated from BM differentiation into a specific lineage or ability to suppress an immune
collected from the iliac crest. The cells are diluted, and the mono- response such as an MLR assay.
nuclear fraction is isolated using a Ficoll-Hypaque density cushion
(GE Healthcare Life Sciences). This fraction is plated into culture
flasks to enrich for adherent cells. The nonadherent population Genetically Modified Cell Therapy Products
is removed after approximately 7 days of incubation. The adherent
cells are washed and detached by incubation with trypsin/ Many cell therapy approaches involve genetic modification of either
ethylenediaminetetraacetic acid (alternatively, TrypZean [Sigma- APCs or effector cells (see earlier discussion). This therapy requires a
Aldrich], a recombinant form of trypsin, can be substituted) and then source of vector that has been manufactured to meet regulatory
cultured and passaged at weekly intervals. In a study designed to requirements. Viral vector specifications changed markedly after the
optimize culture conditions, the best results were obtained when cells death of a gene therapy patient in Philadelphia, and they continue
were cultured in low-glucose Dulbecco’s modified Eagle medium- to evolve. Manufacturers must maintain close contact with the FDA
based media containing 10% fetal bovine serum (human platelet to ensure that their products meet current specifications. Manufactur-
lysate is now frequently substituted) and GlutaMAX (Thermo Fisher) ing and testing of viral vectors is extremely expensive, and use of
instead of L-glutamine. The cells also proliferated better when plated genetically modified products requires additional monitoring of
2
at low cell densities (5000–10,000 cells/cm ) in Falcon flasks. Use of recipients. Use of vectors to transduce or transfect cellular therapy
basic fibroblast growth factor as a growth supplement was found to products ex vivo usually requires additional testing of the product,
be effective; however, it caused HLA-DR induction and upregulated which may include detection of replication-competent virus and
HLA class I expression. This did not affect the immunosuppressive checking the functionality of the introduced vector (by detecting
capabilities of the cells. An increase in osteogenic and adipogenic expression of the gene product).
potential was noted, but neurogenesis and engraftment support for
+
CD34 cells were slightly suppressed. A number of media specifically
designed for culture of MSCs are commercially available (e.g., Gene-Modified Tumor Vaccines
PRIME-XV from Irvine Scientific, STEMPRO MSC SFM, a serum-
free formulation from Invitrogen, and MesenCult, a basal culture Tumor vaccines as an approach to inducing or stimulating immunity
medium from StemCell Technologies). Various investigators have have been evaluated. Various techniques have been tested, including