SUPPLEMENT ARTICLE

Cell therapies for trauma and critical care medicine: critical issues
in translation for cellular and novel therapies in trauma and
critical care

David F. Stroncek,1 Aby J. Mathew,2 and David H. McKenna3

T
he final day of the meeting ended with a session because of their limited clinical efficacy their use was like-
entitled, “Critical Issues in Translation for Cellular wise limited and the cell processing laboratories were rela-
and Novel Therapies in Trauma and Critical tively small. The use of cell therapies has recently begun to
Care.” The goal of this session was to discuss grow; driven to a great extent by the success of genetically
issues critical to the production of cell therapies to be engineered T cells such as CAR T cells for cancer immuno-
utilized in clinical trials and the long-term success and com- therapy.3 The discovery that somatic cells can be repro-
mercialization of such therapies. It was noted that much gramed to embryonic-like stem cells has also increased
progress has been made since the last meeting was held in interest in regenerative medicine. Induced pluripotent stem
2015.1 There are several ongoing clinical trials, and a multi- cells can be manufactured from some CD34+ cells isolated
center Phase II trial of mesenchymal stem cells (MSCs) in from 100 to 200 mL of whole blood. Clinical protocols are
acute lung injury2 was recently completed. Results of this being developed to use iPSC-derived retinal pigment epithe-
trial are much anticipated and should be published shortly. lial cells to treat age-related macular degeneration.4,5 Mes-
While there have been substantial steps forward, there enchymal stromal cells or MSCs are being used in a
remain several challenges ahead. Many of these, number clinical trials for both regenerative medicine and
e.g., regulatory hurdles, timing, dosing, and selection of immunomodulation.
optimal therapy and relevant endpoints, were discussed ear- For many years academic medical centers have been
lier in the meeting. The eight presentations of this session operating cell processing laboratories that processed hema-
focused on three main areas—cell therapy manufacturing topoietic stem cell grafts to support blood and marrow
and testing, biopreservation, and the role of blood centers transplant programs. Some of these cell processing labora-
and professional organizations. The challenges, and perhaps tories have also been manufacturing cell and gene therapies
opportunities, related to cell therapy manufacturing as dis- for use in Phase I and II clinical trials. The growth of MSC
cussed in the final session of the CTTACC meeting are and cancer immunotherapies, particularly CAR T cells, has
resulted in the rapid expansion of cell processing facilities at
summarized here.
many academic centers to support clinical cell and gene
The use of cellular therapies in early phase clinical
therapy programs.
trials is growing rapidly. Cell therapies have been manufac-
Many cell therapies are autologous, and the manufac-
tured at academic medical centers for many years, but
ture of autologous cell therapies presents many unique
challenges. The therapy is tailored to the specific needs of
ABBREVIATION: MSC(s) = mesenchymal stem cell(s)
the patient, and thus every patient requires his or her own
From the 1Department of Transfusion Medicine, Clinical Center, lot of product. The production of these large number of lots
National Institutes of Health, Bethesda, Maryland; 2BioLife requires a considerable amount of in-process and lot
Solutions Inc., Bothell, Washington; and 3Molecular & Cellular release testing, and a considerable amount of labor is
Therapeutics, University of Minnesota, St. Paul, Minnesota. required for quality oversight. The use of autologous cells as
Address reprint requests to: David H. McKenna, Molecular & starting material for cell manufacturing also results in con-
Cellular Therapeutics, University of Minnesota, 1900 Fitch Avenue, siderable variability in the manufacturing process due to
St. Paul, MN 55108; e-mail: [email protected]. variability in the starting cellular material. The variability is
Received for publication February 8, 2018; and accepted April due to genetic variation among individuals, differences in
28, 2018. age and sex, and differences in underlying disease and treat-
doi:10.1111/trf.14832 ment. This variability appears to be especially problematic
© 2019 AABB for the manufacture of CAR T cells to treat patients with
TRANSFUSION 2019;59;854–857 acute lymphocytic leukemia where peripheral blood

854 TRANSFUSION Volume 59, February 2019

 TRANSLATION OF CELLULAR AND NOVEL THERAPIES

lymphocytes are used for the starting material.6,7 Variability ability of MSCs to inhibit lymphocyte proliferation or that
in the starting cellular material appears to be less of a factor measure the release of various cytokines and growth factors.
when manufacturing MSCs since most clinical trials of The goal of biopreservation is to optimize cell yield,
MSCs for immunomodulation and regenerative applications viability, and functionality or potency. The clinical and com-
make use of cells collected from healthy third-party donors. mercial utility of cell therapy products is most certainly
Another challenge of cell therapy manufacturing, often impacted by its stability across all phases of manufacturing,
the case with MSCs, is the need for fetal bovine serum including storage and transport of the source material, in-
(FBS) as a medium supplement to support cell expansion. process steps, and biopreservation of the final cell or tissue
The use of FBS is problematic since it may result in allergic product (frozen or nonfrozen/“fresh”/liquid). Traditional
reactions in the MSC recipient or it could lead to a xenoge- home-brew reagent cocktails (including serum) utilized for
neic infection. In addition, there may be considerable lot- biopreservation may be a point of risk within a cGMP clini-
to-lot variability of FBS, and the supply of FBS is limited.8 cal manufacturing process, and they may be suboptimal
Several studies have found that lysed human platelets options in comparison to preformulated/prepackaged cGMP
(PLTs) can be used as an MSC medium supplement.9 PLT intracellular-like formulations. Therefore, it would be worth-
lysate is now commercially available. Typically, it is derived while to consider best practices recommendations for inte-
from large lots of pooled PLTs to reduce lot-to-lot variabil- grating biopreservation methods within cGMP for cell
ity. While all PLT lysate is manufactured using PLTs from therapy manufacturing, along with consideration to the
screened healthy donors, some groups are investigating the overall quality and regulatory plan.13 Traditionally, biopre-
use of pathogen inactivation technologies with PLT lysate. servation was generally a distinct task for the final product
An obvious barrier to the use of MSCs and other cell (either nonfrozen or frozen), not uncommonly an after-
therapies in clinical trials is the relatively high cost of thought, and optimization might only be explored reactively
expanding these cells. Many clinical trials use relatively from a negative output. Following biopreservation best
large MSC doses and administer the cells on multiple practices recommendations, biopreservation is evaluated or
occasions. Since most MSC products manufactured for validated proactively at each stability risk point, from the
clinical trials can be manufactured using cells from healthy source material through final patient administration of the
third-party donors and cryopreserved MSCs are effective, it cell product.
is possible to manufacture lots of MSCs of sufficient size to When removed from normothermic conditions, cells
treat multiple patients. While the quantity of MSCs that and tissues are vulnerable to various stresses and potential
can be produced by most cell therapy centers based resulting degradation. To mitigate the stability stresses
in academic medical centers is limited,10 at least one throughout the cell manufacturing life cycle, optimization
company has begun to manufacture very large lots of may include focus on (but not limited to): shipping con-
clinical-grade MSCs to reduce the cost of manufacturing tainers, biopreservation media, protocol methods (including
on a per-cell basis.11 These large lots of MSCs can be hypothermic preservation, freezing, thawing, postpreserva-
manufactured in bioreactors using microcarriers. At the tion assessment), and transient warming events. The most
completion of the manufacturing process the MSCs are commonly used protocol methods for cellular therapies and
cryopreserved in standardized quantities using standard regenerative medicine are hypothermic preservation (non-
freezing bags and media. frozen, 2–8 C) and cryopreservation (slow freezing, −1 C/
A final challenge to the clinical application of MSCs is min freezing rate). These methods leverage the decreased
identification of an appropriate measurement of potency. metabolism and reduced cellular degradation that result
Potency is a critical property of all cell therapies since it from low-temperature conditions to preserve biologic integ-
allows for the assessment of consistency among lots and rity and function. However, cellular responses to low tem-
provides a mechanism for assessing the effectiveness of peratures include detrimental outcomes, such as disruption
each cell therapy lot. Assessing the potency of MSCs is par- of ionic and osmotic balance, accumulation of free radicals
ticularly difficult because of their heterogeneity and the very and reactive oxygen species, physical damage from ice for-
wide range of cytokines and growth factor that they pro- mation, chemical damage from solute toxicity, and multiple
duce. Further, the characteristics defining clinical efficacy of mechanisms of cell death (necrosis, apoptosis, secondary
MSCs are not completely understood, and in some cases necrosis).13 Understanding these outcomes and the role of
multiple properties are likely involved.12 Animal models for delayed-onset cell death13 in each cell manufacturing prod-
assessment of potency of MSCs for many clinical applica- uct is critical, as measurement and interpretation of viability
tions have been described; however, due to limits of lot size can be complex. Often the assessment involves consider-
of MSCs, assessment of potency with animal models ation of “perceived viability” immediately after preservation
remains impractical for most clinical trials. in vitro assays versus long-term “true viability” of the cell product, as post-
are often used to assess potency, and a number of assays preservation cellular mechanisms may be slower to initiate,
are being developed and investigated as potency assays for manifest, and resolve. The mechanisms related to apoptosis,
MSCs. These include standardized assays that measure the necrosis, and secondary necrosis often take 24 to 48 hours

Volume 59, February 2019 TRANSFUSION 855

STRONCEK ET AL.

after preservation (e.g., on thaw) to manifest, before nor- through screening (i.e., donor health questionnaire) and
malization of viable cell recovery and return of function. infectious disease testing; they collect whole blood and aphe-
Depending on the methods utilized for cell assessment, and resis products, and several centers perform varying levels of
time points of assessment after preservation, there may be cell processing. Additionally, blood centers have processes
significant pitfalls in understanding the perceived viability and procedures established for storage and distribution, criti-
versus true viability of the cell product. cal steps before patient administration. Finally, some blood
Beyond the utility of the biopreservation methods for centers also provide specialty services such as HLA typing
cell yield, viability, and function, it is also necessary to align and CD34+ cell enumeration, and a few have basic science
the biopreservation tools with the appropriate quality and and translational research laboratories. A handful of blood
regulatory footprint for cGMP clinical cell manufacturing. centers, such as the New York Blood Center, have established
Within cellular therapies and regenerative medicine applica- licensed cord blood banks.
tions, biopreservation media are ancillary materials in the Professional organizations or societies also hold great
cell manufacturing process, not medical devices or drugs potential for contribution to the field. While most such orga-
requiring stand-alone approvals or marketing authoriza- nizations are focused heavily on their educational mission
tions. They may also be qualified for excipient usage by the and networking opportunities, some such as AABB and the
cell therapy developer. As with other raw materials utilized International Society for Cellular Therapy (ISCT), participate
within cell and tissue manufacturing, biopreservation tools heavily in advocacy on behalf of society members and other
are recommended to be appropriately qualified for func- stakeholders with the Food and Drug Administration. Both
tional utility and risk mitigation, dependent on the needs of AABB and the Foundation for the Accreditation of Cellular
each clinical product and application. Therapies (FACT), a nonprofit corporation co-founded by
In consideration of biopreservation best practices, it ISCT and the American Society of Blood and Marrow Trans-
would be recommended to optimize biopreservation plantation (ASBMT), maintain voluntary accreditation pro-
methods and mitigate negative impact on stability, from grams in cellular therapy. One international group, the
source material through final patient administration. At Biomedical Excellence for Safer Transfusion (BEST) collabo-
each point in the manufacturing life cycle that is vulnerable rative, includes a cell therapy team that focuses on the
to stability risk, a gap analysis can allow for understanding assessment of current practices in the collection,
the impact. Methods for stability improvement may include manufacturing, testing, storage, and shipment of cell ther-
(but are not limited to): utilizing intracellular-like media apy products.17,21,22 Cell therapy-focused professional socie-
designed for low-temperature preservation (rather than ties have increased in number over the past several years,
isotonic-based media) and enhanced control of ionic bal- and many have expanded their offerings to their members.
ance, osmotic stabilization, pH buffering, and free radical There appears to be duplicative effort on some fronts, and
scavenging; mitigation of cell death stress activation during the sustained existence of these organizations will rely on
and after preservation; transition to tools and reagents man- their continued relevance to their membership.
ufactured per cGMP with ability to integrate with a clinical Standards are requirements based upon “good medical
quality and regulatory footprint; optimization of freezing practice, scientific data, principles associated with good
rates and thawing methods; and avoidance of damaging manufacturing practices and quality assurance, and applica-
thermal cycling or transient warming events, including dur- ble regulations.”23 The decision to meet these requirements
ing transport. Optimized protocols14 utilizing qualified, and become accredited is entirely voluntary. However, the
cGMP, serum-free, protein-free biopreservation media have standards program as established by AABB, as well as FACT,
demonstrated improved stability of source material, in- offers a pathway for an institutional cell therapy program to
process intermediates, and final product and have also been attain best practices and compliance with the expectations
qualified for excipient application without wash after preser- of regulators. The eighth edition of the AABB Cell Therapy
vation (HypoThermosol FRS/CryoStor, BioLife Solutions, Standards was published in May 2017 and covers activities
Inc.).15–20 related to hematopoietic progenitor cells, umbilical cord
While most MSCs and other cell therapies to date have blood banks, and somatic cells (e.g., MSCs, pancreatic
been manufactured in academic medical centers or contract islets). These standards are written by a committee of tech-
manufacturing organizations, blood centers have had rela- nical experts, liaisons from other AABB committees, and
tively limited involvement with cell therapies. There is, in representatives from other organizations and are revised on
fact, great potential for a higher level of involvement by a 24-month cycle. FACT uses a similar approach and cur-
blood centers. While blood centers have traditionally only rently has a sixth edition of their International Standards for
collected and processed blood and blood components Hematopoietic Cellular Therapy Product Collection, Proces-
(e.g., PLTs, red blood cells), some are well suited for provid- sing, and Administration (2015) and a first edition of their
ing cell therapies because of their existing infrastructure and Standards for Immune Effector Cells (2017).
expertise (i.e., technical, quality/regulatory, and medical/ While there has clearly been much progress in the
scientific). Blood centers routinely determine donor eligibility application of cellular therapies in trauma and critical care

856 TRANSFUSION Volume 59, February 2019

 TRANSLATION OF CELLULAR AND NOVEL THERAPIES

over the past several years, there remain challenges in 11. Cunha B, Aguiar T, Carvalho SB, et al. Bioprocess integration
manufacturing. These include donor and lot-to-lot variabil- for human mesenchymal stem cells: from up to downstream
ity, cost and scalability, and proper characterization and processing scale-up to cell proteome characterization. J Bio-
assessment of the final product. Optimization of biopreser- technol 2017;248:87–98.
vation, including nonfrozen or liquid storage and cryopres- 12. Mendicino M, Bailey AM, Wonnacott K, et al. MSC-based prod-
ervation, is a critical need in manufacturing and is uct characterization for clinical trials: an FDA perspective. Cell
paramount to the long-term success and accessibility of Stem Cell 2014;14:1415.
therapies. Blood centers and professional organizations offer 13. Hawkins B, Abazari A, Mathew AJ. Biopreservation best prac-
expertise and experience that will contribute greatly to the tices for regenerative medicine GMP manufacturing & focus on
advancement of the field. optimized biopreservation media. Cell Gene Therapy Insights
2017;3:345–58.
14. Ramos TV, Mathew AJ, Thompson ML, et al. Standardized
REFERENCES cryopreservation of human primary cells. Curr Protoc Cell Biol
1. Pati S, Pilia M, Grimsley JM, et al. Cellular therapies in trauma 2014;64:A.3I.1–8.
and critical care medicine: Forging new frontiers. Shock 2015; 15. Lu TL, Pugach O, Somerville R, et al. A rapid cell expansion
44:505–23. process for production of engineered autologous CAR-T cell
2. Liu KD, Wilson JG, Zhuo H, et al. Design and implementation therapies. Human Gene Therapy Methods 2016;27:209–18.
of the START (STem cells for ARDS Treatment) trial, a phase 16. Brown C, Alizadeh D, Starr R, et al. Regression of glioblastoma
1/2 trial of human mesenchymal stem/stromal cells for the after chimeric antigen receptor T-cell therapy. N Engl J Med
treatment of moderate-severe acute respiratory distress syn- 2016;375:2561–9.
drome. Ann Intensive Care 2014;4:22. 17. Worsham DN, Reems JA, Szczepiorkowski ZM, et al. Clinical
3. Hartmann J, Schüßler-Lenz M, Bondanza A, et al. Clinical methods of cryopreservation for donor lymphocyte infusions
development of CAR T cells-challenges and opportunities in vary in their ability to preserve functional T cell subpopula-
translating innovative treatment concepts. EMBO Mol Med tions. Transfusion 2017;57:1555–65.
2017;9:1183–97. 18. Kunkele A, Taraseviciute A, Finn L, et al. Preclinical assessment
4. Nazari H, Zhang L, Zhu D, et al. Stem cell based therapies for of CD171-directed car t-cell adoptive therapy for childhood
age-related macular degeneration: the promises and the chal- neuroblastoma: CE7 epitope target safety and product
lenges. Prog Retin Eye Res 2015;48:1–39. manufacturing feasibility. Clin Cancer Res 2017;23:466–7.
5. Song MJ, Bharti K. Looking into the future: using induced plu- 19. Whitehouse M, Howells N, Parry M, et al. Repair of torn
ripotent stem cells to build two and three dimensional ocular avascular meniscal cartilage using undifferentiated autolo-
tissue for cell therapy and disease modeling. Brain Res 2016; gous mesenchymal stem cells: from in vitro optimization to
1638(Pt A):2–14. a first-in-human study. Stem Cells Transl Med 2017;6:
6. Stroncek DF, Ren J, Lee DW, et al. Myeloid cells in peripheral 1237–48.
blood mononuclear cell concentrates inhibit the expansion of 20. Bartunek J, Behfar A, Dolatabadi D, et al. Cardiopoietic stem
chimeric antigen receptor T cells. Cytotherapy 2016;18:893–901. cell therapy in heart failure. J Am Coll Cardiol 2013;61:
7. Stroncek DF, Lee DW, Ren J, et al. Elutriated lymphocytes for 2329–38.
manufacturing chimeric antigen receptor T cells. J Transl Med 21. Liu S, de Castro LF, Jin P, et al. Manufacturing differences
2017;15:59. affect human bone marrow stromal cell characteristics and
8. Brindley DA, Davie NL, Culme-Seymour EJ, et al. Peak serum: function: comparison of production methods and products
implications of serum supply for cell therapy manufacturing. from multiple centers. Sci Rep 2017;27:46731.
Regen Med 2012;7:7–13. 22. Gniadek TJ, Garritsen HS, Stroncek D, et al. Optimal Storage
9. Strunk D, Lozano M, Marks DC, et al. International forum on Conditions for Apheresis Research (OSCAR): a Biomedical
GMP-grade human platelet lysate for cell propagation: sum- Excellence for Safer Transfusion (BEST) collaborative study.
mary. Vox Sang 2018;113:80–7. Transfusion 2018;58:461–9.
10. Sabatino M, Ren J, David-Ocampo V, et al. The establishment 23. Standards setting [Internet]. Bethesda: AABB; 2018 [cited 2018
of a bank of stored clinical bone marrow stromal cell products. July 27]. Available from: www.aabb.org/sa/standards/Pages/
J Transl Med 2012;10:23. default.aspx.

Volume 59, February 2019 TRANSFUSION 857