Multicenter cell processing for cardiovascular regenerative medicine applications: the Cardiovascular Cell Therapy Research Network (CCTRN) experience

Abstract

Abstract Background aims. Multicenter cellular therapy clinical trials require the establishment and implementation of standardized cell-processing protocols and associated quality control (QC) mechanisms. The aims here were to develop such an infrastructure in support of the Cardiovascular Cell Therapy Research Network (CCTRN) and to report on the results of processing for the first 60 patients. Methods. Standardized cell preparations, consisting of autologous bone marrow (BM) mononuclear cells, prepared using a Sepax device, were manufactured at each of the five processing facilities that supported the clinical treatment centers. Processing staff underwent centralized training that included proficiency evaluation. Quality was subsequently monitored by a central QC program that included product evaluation by the CCTRN biorepositories. Results. Data from the first 60 procedures demonstrated that uniform products, that met all release criteria, could be manufactured at all five sites within 7 h of receipt of BM. Uniformity was facilitated by use of automated systems (the Sepax for processing and the Endosafe device for endotoxin testing), standardized procedures and centralized QC. Conclusions. Complex multicenter cell therapy and regenerative medicine protocols can, where necessary, successfully utilize local processing facilities once an effective infrastructure is in place to provide training and QC.

Figures

Figure 1

Analysis of Sixty Sepax Clinical Separations The two columns to the left of the graph show the numbers of total nucleated cells and red cells (TRC) in the product as measured by automated cell counters. The next two columns show these results expressed as percent recoveries of the initial numbers of these cells loaded onto the Sepax. The three columns to the right of the graph show the three part differential on the products as measured by automated cell counter at three centers, by flow cytometry at one center and manually at the last center. All numbers shown are mean ± 1 standard deviation. Standard deviation on %TRC depletion is too small to show.

Figure 2

Sepax Processing Results shown by Processing Laboratory The bars to the left of the graph show the mean numbers of nucleated marrow cells harvested at each clinical center. The bars in the center show the mean percent recovery of nucleated cells in the Sepax products. The bars to the right show the mean percent depletion of red cells in the Sepax products obtained at each center. Error bars show 1 standard deviation.

Figure 3

Recovery of Progenitor Cell Populations in Sepax Products Progenitor cell recovery was measured at one processing center. The bars to the left show the mean numbers of and CD34- and CD133 positive cells in the Sepax products. To the right are the percent recoveries of these cells together with the recovery of total nucleated cells. Error bars show 1 standard deviation.

Figure 4

Quality Control Test Results on Final Cell Products Shown are the results of release testing of the final cellular therapy products. All products met the release criteria of viability ≥ 70% by Trypan blue exclusion, sterility by Gram stain, and endotoxin ≤ 5.0 EU/kg. Additional test results for CFU- growth and viability by 7-AAD are shown. CFU growth was seen in all samples plated according to SOP, however, in one case too few cells were plated and colonies were not detected, and in a second the assay was omitted in error.

Figure 5

Characteristics of Products prepared for the TIME/late Time Protocol Shown are the mean numbers of nucleated, red and progenitor cells. For the progenitor populations the bars to the left show numbers based on flow cytometric analysis performed at the processing centers; bars to the right show numbers based on analysis perfomed at the Minnesota biorepository. Error bars show 1 standard deviation.

Figure 6

Characteristics of Products prepared for the FOCUS Protocol Shown are the mean numbers of nucleated, red and progenitor cells. For the progenitor populations the bars to the left show numbers based on flow cytometric analysis performed at the processing centers; bars to the right show numbers based on analysis perfomed at the Minnesota biorepository. Error bars show 1 standard deviation.