Human Umbilical Cord Derived Mesenchymal Stem Cell Sheets for Clinical Use

Manufacture and Quality Control of Human Umbilical Cord Derived Mesenchymal Stem Cell Sheets for Clinical Use

Umbilical Cord-Derived Mesenchymal Stem Cells and Sheet Technology

Mesenchymal stem cells (MSCs) are multipotent stem cells that have vast therapeutic potential due to their ability to differentiate into multiple different cell types, modulate the immune system, and self-renew. MSCs are being used in clinical trials for treatment of many diseases like Crohn's Disease, myocardial infarction, myocardial ischemia, burns, and leukemia, which currently have no cure.¹ Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are frequently being used as a source for stem cells because they can be obtained via noninvasive collection methods, have low immunogenicity, and have lower levels of senescence and background disease compared to adult stem cells.^2,3

Cell sheet technology improves mesenchymal stem cell transplantation efficiency by increasing the quantity of cells delivered to site and improving cell viability. Cell sheet technology also increases the functionality of the stem cells by enhancing the metabolic environment,2 improving paracrine factor delivery, and increasing expression of cell interaction proteins and regenerative proteins (e.g., vascular endothelial growth factor, hepatocyte growth factor, IL-10).⁴

Advancements in the growth and preservation conditions need to be made to improve patient safety thereby increasing the therapeutic potential of UC-MSCs. Laboratory growth conditions include components like fetal bovine serum and exogenous growth factors, which present safety risks including potential allergic reactions and carcinogenic risks to patients. These components are unsuitable for clinically safe and therapeutically relevant cell sheet preparations, meaning researchers must develop new growth media and cell sheet preservation methods to meet Good Manufacturing Practice (GMP) regulations and thus be useable for clinical applications.⁵

Modified growth media and preservation reagents for improved patient safety.

The researchers focused on using components from approved therapeutics to develop a modified growth media and a Petri dish coating matrix. The final results were a growth media made of only basic cell culturing media (α-MEM) supplemented with 0.1% human serum albumin (HSA) and a coating matrix with 10ng/mL of fibrinogen. Using these new medias, samples from eight individuals were used to fabricate UC-MSC sheets. All the samples resulted in successful generation of UC-MSC sheets with over 85% viability.⁵

After the successful determination of cell sheet production reagents, preservation methods had to be established. Three different preservation solutions were investigated: normal saline with 1% HSA, a commercial preservation solution that is classified as a category III medical device, and Hypothermosol from Biolife Solutions. Post-24-hour incubation in the three solutions, UC-MSC sheets were examined for sheet integrity, viability, and cell number. The normal saline with 1% HSA had the poorest results out of the three as there was significant sheet damage and the most loss in cell viability. While the commercial preservative and Hypothermosol had very similar results, the commercial preservative was selected for use, since it is a class III medical device; its ingredients were of lower safety and compliance risk.⁵

Modified growth media and preservation reagents for improved patient safety.

The researchers focused on using components from approved therapeutics to develop a modified growth media and a Petri dish coating matrix. The final results were a growth media made of only basic cell culturing media (α-MEM) supplemented with 0.1% human serum albumin (HSA) and a coating matrix with 10ng/mL of fibrinogen. Using these new medias, samples from eight individuals were used to fabricate UC-MSC sheets. All the samples resulted in successful generation of UC-MSC sheets with over 85% viability.⁵

After the successful determination of cell sheet production reagents, preservation methods had to be established. Three different preservation solutions were investigated: normal saline with 1% HSA, a commercial preservation solution that is classified as a category III medical device, and Hypothermosol from Biolife Solutions. Post-24-hour incubation in the three solutions, UC-MSC sheets were examined for sheet integrity, viability, and cell number. The normal saline with 1% HSA had the poorest results out of the three as there was significant sheet damage and the most loss in cell viability. While the commercial preservative and Hypothermosol had very similar results, the commercial preservative was selected for use, since it is a class III medical device; its ingredients were of lower safety and compliance risk.⁵

Establishment of Large-Scale UC-MSC Sheet Production

Since the long-term vision is to manufacture UC-MSC cell sheets for therapeutic purposes, quality standards were developed involving product characteristics, safety, and functionality. All of these processes were managed in accordance with GMP regulations. The two-tiered cell banks including the master cell bank (MCB) and working cell bank (WCB) were established as intermediate products for final cell sources for UC-MSC sheet production. MCBs were used to establish WCBs, which were then used to generate UC-MSC sheets. According to the established processes, one umbilical cord could be used to establish one batch of MCB; one batch of MCB could be used to establish several batches of WCBs; one batch of WCBs could be used to produce several batches UC-MSC sheet products. In testing these processes, three batches of MCBs with over 300 million cells in each were successfully established from three umbilical cords. Three WCBs were established with over 2.4 billion cells in each, from three independent MCBs. Subsequently, three batches of UC-MSC sheet products were successfully generated from three independent WCB batches. Thirty-five sheets were produced in each batch – enough to be used for at least ten patients.⁵

UC-MSC batches retain stem cell properties and functionality.

All of the MCB and WCB batches met the established quality standards for surface markers, viability, and density. The UC-MSCs were able to be differentiated into osteoblasts, chondroblasts, and adipocytes. These stem cells also had immunomodulatory effects through secretion of cytokines like hepatocyte growth factor, IL-6, IL-8, and TNF-α. Additionally, the three MCB and WCB batches were found to have no microbe contamination, no cross-cell contamination, and no tumorigenicity.⁵

The batches of UC-MSC sheet products were examined immediately after production and after a 24-hour incubation in the preservative solution. Fresh and preserved samples maintained cell integrity with over 70% viability and less than 15% apoptosis. The stem cells were positive (≥ 95%) for stem cells markers CD73, CD90, and CD105, and negative (positive cell proportion ≤ 2%) for markers CD11b, CD19, CD34, CD45, and HLA-DR. Similar to the UC-MSCs derived from the MCBs and WCBs, the UC-MSC sheets could be induced to differentiate into chondroblasts, osteoblasts, and adipocytes. All three batches of UC-MSC sheets, whether fresh or preserved, satisfied the quality standards which included assessing high risk substances like bovine serum albumin, human serum albumin, gentamicin, fibrinogen, basic fibroblast growth factor, and TrypLE via ELISAs. All the UC-MSC sheets also secreted cytokines similar to the MCBs and WCBs.⁵

This study developed and optimized new reagents and systems for producing and preserving UC-MSC sheets at large scale. These strategies increased the therapeutic potential of UC-MSC sheets by decreasing patient risks, increasing feasibility, and maintaining cell viability and functionality.