The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens.

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

Financial analysis of large-volume delayed sampling to reduce bacterial contamination of platelets.

BACKGROUND: Effective and financially viable mitigation approaches are needed to reduce bacterial contamination of platelets in the US. Expected costs of large-volume delayed sampling (LVDS), which would be performed by a blood center prior to shipment to a hospital, were compared to those of pathogen reduction (PR), point-of-release testing (PORt), and secondary bacterial culture (SBC). METHODS: Using a Markov-based decision-tree model, the financial and clinical impact of implementing all variants of LVDS, PR, PORt, and SBC described in FDA guidance were evaluated from a hospital perspective. Hospitals were assumed to acquire leukoreduced apheresis platelets, with LVDS adding $30 per unit. Monte Carlo simulations were run to estimate the direct medical costs for platelet acquisition, testing, transfusion, and possible complications associated with each approach. Input parameters, including test sensitivity and specificity, were drawn from existing literature and costs (2018US$) were based on a hospital perspective. A one-way sensitivity analysis varied the assumed additional cost of LVDS. RESULTS: Under an approach of LVDS (7-day), the total cost per transfused unit is $735.78, which falls between estimates for SBC (7-day) and PORt. Assuming 20,000 transfusions each year, LVDS would cost $14.72 million annually. Per-unit LVDS costs would need to be less than $22.32 to be cheaper per transfusion than all other strategies, less than $32.02 to be cheaper than SBC (7-day), and less than $196.19 to be cheaper than PR (5-day). CONCLUSIONS: LVDS is an effective and cost-competitive approach, assuming additional costs to blood centers and associated charges to hospitals are modest.

A simple and cost-efficient adherent culture platform for human gastric primary cells, as an in vitro model for Helicobacter pylori infection.

BACKGROUND: Most two- dimensional in vitro models for studying host- H. pylori interactions rely on tumor-derived cell lines, which harbor malignant alterations. The recent development of human gastric organoids has overcome this limitation and provides a highly sophisticated, yet costly, short-term model for H. pylori infection, with restricted use in low-budget centers. METHOD: Tissue specimens from upper, middle, and lower stomachs of H. pylori-negative volunteers were collectively dispersed and cultured on mouse embryonic fibroblast (MEF) or collagen-coated plates. Gastric primary cells (GPCs) were evaluated by light microscopy, immunostaining, qRT-PCR and ELISA analysis of cellular secretions, before and after H. pylori infection. RESULTS: The formation and long-term (up to 1 year) maintenance of GPCs was highly dependent on adherent inactivated MEF cells, cultured in enriched media. These cells were multipassageable and able to undergo stable freezer storage and subsequent revival. The cellular composition of GPCs included the combination of cytokeratin 18 (CK18) and E-cadherin (E-cad)-positive epithelial cells, MUC5AC-positive gastric cells, and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)-positive progenitor cells. These cells produced significant amounts of gastric pepsinogens I and II. GPCs also allowed for extended (up to 96 hours) H. pylori infection, during which they underwent morphological alterations (cellular vacuolation and elongation) and hyperproduction of gastric pepsinogens and inflammatory cytokines (IL-8 and TNF-α). CONCLUSION: We, hereby, present a simple, consistent, and cost-efficient gastric cell culture system, which provides a suitable model for extended in vitro infection of H. pylori. This platform can be employed for a variety of gastric-related research.

A refined rat primary neonatal microglial culture method that reduces time, cost and animal use.

BACKGROUND: Primary microglial cultures have been used extensively to facilitate the development of therapeutic strategies for a variety of CNS disorders including neurodegeneration and neuropathic pain. However, existing techniques for culturing these cells are slow and costly. NEW METHOD: Here, we report a refined protocol based on our previously published methods described by Clark et al., which reduces in the time, reagents and the number of animals used for each culture whilst yielding high number and excellent quality microglial cells. RESULTS: Our refined protocol offers an isolation of >96% microglia from a mixed glial culture after only four days of incubation. It results in a high yield of microglia, in excess of one million cells per cortex with predominantly resting morphology and a low level of cell activation. COMPARISON WITH EXISTING METHOD(S): Compared to conventional procedures our refined protocol requires only one third of the time to prepare high quality microglial cultures, cuts the cost more than four-fold, and significantly reduces the number of animals used per culture. CONCLUSION: Our consistent, reliable, and time/cost effective microglial culture protocol is crucial for efficient in vitro screening of potential therapeutics. By dramatically reducing the culture time from 2 weeks to just 4 days and increasing the laboratory research output it has implications for the Reduction, Refinement and Replacement policies endorsed by many government funding agencies and animal research regulatory bodies.

Process cost and facility considerations in the selection of primary cell culture clarification technology.

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.