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Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

ABSTRACT: Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1-3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

SUBMITTER: MacDonald KN

PROVIDER: S-EPMC7474528 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

MacDonald Katherine N KN Ivison Sabine S Hippen Keli L KL Hoeppli Romy E RE Hall Michael M Zheng Grace G Dijke I Esme IE Aklabi Mohammed Al MA Freed Darren H DH Rebeyka Ivan I Gandhi Sanjiv S West Lori J LJ Piret James M JM Blazar Bruce R BR Levings Megan K MK

Cytotherapy 20191203 12

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manuf ...[more]

PMID: 31810768

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Project description:ObjectiveCritical process parameters (CPPs) identification is an important step of the implementation of quality by design (QbD) concept. There are many CPP identification methods, such as risk analysis method, sensitivity analysis method, multiple linear regression method, standard partial regression coefficient (SPRC) method, and so on. The SPRC method can consider multiple process critical quality attributes (CQAs) simultaneously, but the determination of CPP number is subjective. Therefore, new CPP identification method is still required.MethodsThe manufacturing process of Astragali Radix extract, which contained water reflux extraction, concentration, and ethanol precipitation, was used as an example. First, the multiple process CQAs were determined to be the yield of pigment, dry matter, sugars, and active ingredients. Second, the potential CPPs were determined by a knowledge organization method. Plackett-Burman designed experiments were then performed. A weighted determination coefficient ( Rw2 ) method was presented to identify CPPs. In this method, the importance of different CQAs was considered. Process parameters were removed one-by-one according to their importance index. The decrease in Rw2 was used to characterize the importance of the removed parameter. If the decrease of Rw2 was less than a preset threshold, the removed parameter was not a CPP.ResultsDuring the manufacturing process of Astragali Radix extract, the potential CPPs determined by the knowledge organization method were water consumption, reflux extraction time, extraction frequency, ethanol content, ethanol consumption, and concentration endpoint. Reflux extraction time, the first ethanol consumption, the second ethanol consumption, and the second ethanol precipitation refrigeration temperature were found to be CPPs using the weighted determination coefficient method with the threshold of 10%.ConclusionUsing the weighted determination coefficient method, CPPs can be determined with all the CQAs considered based on their importance. The determination of CPP number is more objective compared with the SPRC method.

Project description:The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography–mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3′,5′-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

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Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

Publications

Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol.

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