Local manufacturing processes contribute to variability in human mesenchymal stromal cell expansion while growth media supplements contribute to variability in gene expression and cell function: a Biomedical Excellence for Safer Transfusion (BEST) collaborative study.

Background Aims: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media.

Methods: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

Background: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay.

Study Design And Methods: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories.

Acellular human amniotic fluid protects the ischemic-reperfused rat myocardium.

Amniotic products are potent immunomodulators used clinically to repair tissue injury. Little information exists regarding the potential of cell-free human amniotic fluid (hAF) to treat cardiovascular disease. Herein, we sought to determine the influence and efficacy of acellular hAF on myocardial ischemia-reperfusion injury.

Red Cell Depletion of Major ABO Incompatible Bone Marrow Hematopoietic Progenitor Cells HPC(M) with the Spectra Optia®.

Objective: Major ABO incompatible hematopoietic progenitors from bone marrow (HPC(M)) donor collections that are destined for clinical transplantation are typically processed to deplete products of red blood cells (RBCs). The purpose of this study was to compare RBC depletion when using the Spectra Optia® relative to a 2-step method involving a COBE2991 instrument to obtain a buffy coat followed by a hydroxyethyl starch (HES) density gradient (COBE+HES) of the buffy coat.

Methods: Post-processing recoveries of products undergoing 4, 8, and 10 bone marrow processing (BMP) cycles (i.

A multicenter evaluation of heterogeneity in cellular therapy processing laboratory procedure times to assess workload capacity.

Background: Growth in size and complexity of clinical hematopoietic progenitor cell (HPC) transplant programs necessitates parallel increases in cellular therapy laboratory (CTL) workload. Typically individually developed, HPC product processing is labor and time intensive. Variation in procedure type and numbers across CTLs complicates direct comparisons, and benchmark data are not readily available.

Human Mesenchymal Stromal Cell (MSC) Characteristics Vary Among Laboratories When Manufactured From the Same Source Material: A Report by the Cellular Therapy Team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

Background: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods and different source materials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material.

Methods And Methods: Five aliquots from each of three different BM donors were distributed to five independent laboratories.

Variations in novel cellular therapy products manufacturing.

Background Aims: At the frontier of transfusion medicine and transplantation, the field of cellular therapy is emerging. Most novel cellular therapy products are produced under investigational protocols with no clear standardization across cell processing centers. Thus, the purpose of this study was to uncover any variations in manufacturing practices for similar cellular therapy products across different cell processing laboratories worldwide.

Processed human amniotic fluid retains its antibacterial activity.

Background: Human amniotic fluid (AF) contains numerous nutrients, trophic factors and defense proteins that provide a nurturing and protective environment for fetal development. Based on reports that AF has antibacterial, anti-inflammatory and regenerative properties, we designed a novel method to process AF for use in clinical care.

Methods: Six randomly selected lots of processed AF (pAF) were examined to determine whether they retained their antibacterial activity against a panel of wound-associated pathogens E.

Current practices for viability testing of cryopreserved cord blood products: an international survey by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

Background: Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility.

Study Design And Methods: A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally.

Microvasculature-directed thrombopoiesis in a 3D in vitro marrow microenvironment.

Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets.

Comparative analyses of industrial-scale human platelet lysate preparations.

Background: Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product.

Study Design And Methods: Nine industrial-scale, serum-based PL manufacturing runs (i.

Clinical methods of cryopreservation for donor lymphocyte infusions vary in their ability to preserve functional T-cell subpopulations.

Background: Cryopreserved donor lymphocyte infusion (DLI) products are manufactured and administered to treat relapse after allogeneic hematopoietic stem cell transplantation. Reported clinical responses to DLIs vary broadly, even within the same group of patients. While there is an implicit recognition of the fact that different manufacturing protocols may have specific effects on different cell types, cryopreservation protocols are frequently derived from our experience in the cryopreservation of stem cell products and do not account for the heterogeneous functional nature of DLI T-cell populations.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods.

Collection and characterization of amniotic fluid from scheduled C-section deliveries.

Amniotic fluid (AF) possesses anti-inflammatory, anti-microbial and regenerative properties that make it attractive for use in clinical applications. The goals of this study were to assess the feasibility of collecting AF from full-term pregnancies and to evaluate non-cellular and cellular properties of AF for clinical applications. Donor informed consent and medical histories were obtained from pregnant women scheduled for C-sections and infectious disease testing was performed the day of collection.

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products.

Background Aims: The question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.

Methods: Cryovials (n = 233) frozen for an average of 6.

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

Background Aims: Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures.

Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

Background Aims: Wide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility.

Methods: A survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed.

Expression of plasma membrane receptor genes during megakaryocyte development.

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development.

Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition.

Background: Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%.

A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures.

Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs.

A journey to produce platelets in vitro.

Allogeneic platelet transfusions protect patients from bleeding episodes and also make aggressive medical procedures such as those involving marrow transplants requiring chemotherapy and/or radiotherapy possible. These patients are dependent upon an unfailing supply of platelets that can sometimes be in short supply due to high demands coupled with an extremely short expiration date for platelet products of only 5 days. One approach that is under investigation to overcome platelet shortages is to harness the extraordinary capabilities of stem cells to proliferate and differentiate into various cell types and to use this ability to specifically produce clinical scale quantities of functional platelets in bioreactors.

Distinct functional effects for dynamin 3 during megakaryocytopoiesis.

Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs.