Variability in subjective review of umbilical cord blood colony forming unit assay

Powell, Kimerly; Kwee, Edward; Nutter, Benjamin; Herderick, Edward E.; Paul, Philip; Thut, Dawn; Boehm, Cynthia; Muschler, George F.

doi:10.1002/cyto.b.21376

Cited by 12 publications

(5 citation statements)

References 13 publications

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“…Naturally, better quality regressions arose from more quantitative measures, such as FI TNC number ( Table 2 ). Both CFU and CAFC outputs suffer from some subjectivity, inherent to these specific assays, requiring a significant dependence on experimental technique to originate results that make modeling possible ( Purton and Scadden, 2007 ; Powell et al, 2016 ). The existence of a single outlier proves the quality of the response variable measurements.…”

Section: Discussionmentioning

confidence: 99%

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

Branco

Bucar

Moura-Sampaio

et al. 2020

Front. Bioeng. Biotechnol.

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Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34 + cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34 + and CD34 + CD90 +) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

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Section: Discussionmentioning

confidence: 99%

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

Branco

Bucar

Moura-Sampaio

et al. 2020

Front. Bioeng. Biotechnol.

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“…Thus, this is the first application of the ASTM Standard Method F‐2944‐12 to the challenge of quantitative automated CTP‐C assay . This Standard is important because it enables standardized and repeatable measurements within a lab and reproducible between labs, advancing the field from its historic dependence on highly subjective manual methods, which are known to vary within and between human observers …”

Section: Discussionmentioning

confidence: 99%

Progenitor cells from different zones of human cartilage and their correlation with histopathological osteoarthritis progression

Mantripragada

Bova

Boehm

et al. 2017

Journal Orthopaedic Research

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Cell-based therapies development for the treatment of osteoarthritis (OA) requires an understanding of the disease progression and attributes of the cells resident in cartilage. This study focused on quantitative assessment of the concentration and biological potential of stem and progenitor cells resident in different zones of cartilage displaying macroscopic Outerbridge grade 1-2 OA, and their correlation with OA progression based on established histologic scoring system. Lateral femoral condyles were collected from 15 patients with idiopathic OA and varus knees undergoing total knee arthroplasty. Superficial(C , top ∼ 500 µm) and deep cartilage(C ) was separated. Chondrogenic Connective Tissue Progenitors (CTP-C) were assayed by standardized Colony-Forming-Unit assay using automated image analysis (Colonyze ) based on ASTM standard F-2944-12. Cell concentration (cells/mg) was significantly greater in C (median: 7,000; range: 3,440-17,600) than C (median: 5,340; range: 3,393-9,660), p = 0.039. Prevalence (CTPs/million cells) was not different between C (median: 1,274; range: 0-3,898) and C (median:1,365; range:0-6,330), p = 0.42. In vitro performance of CTP-C progeny varied widely within and between patients, manifest by variation in colony size and morphology. Mean histopathological Mankin score was 4.7 (SD = 1.2), representing mild to moderate OA. Tidemark breach by blood vessels was associated with lower C cell concentration (p = 0.02). Matrix degradation was associated with lower C cell and CTP-C concentration (p = 0.015 and p = 0.095, respectively), independent of articular surface changes. These findings suggest that the initiation of OA may occur in either superficial or deep zones. The pathological changes affect CTP-Cs in C and C cartilage zones differently. The heterogeneity among the available CTP-Cs in C and C suggests performance-based selection to optimize cell-sourcing strategies for therapy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1728-1738, 2018.

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“…These quantitative methods have been adopted in accordance with principles articulated as an ASTM Standard Method for ''Automated Colony Forming Unit (Assays-Image Acquisition and Analysis Method for Enumerating/Characterizing Cells and Colonies in Culture-ASTM # F2944-12). Using this quantitative, standardizable, and reproducible automated tool will help overcome limitations associated with manual methods for cell processing including cell and colony counting as well as colony selection, which are extremely time-consuming, inaccurate, and inconsistent within and between users (Powell et al 2016(Powell et al , 2007.…”

Section: Automated Lfov Cell and Colony Analysis-colonyze Tmmentioning

confidence: 99%

Automated in-process characterization and selection of cell-clones for quality and efficient cell manufacturing

et al. 2020

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Delivery of safe, effective and reliable cellular therapies, whether based on mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs), demand standardization of cell culture protocols. There is a need to develop automation platform that enables the users to generate culture expanded human cell populations that improves the quality and reduces batch-to-batch variation with respect to biological potential. Cell X TM robot was designed to address these current challenges in the cell fabrication industry. It utilizes non-invasive large field of view quantitative image analysis to guide an automated process of targeted ''biopsy'' (cells or media), ''picking'' (selection) of desired cells or colonies, or ''weeding'' (removal) of undesired cells, thus providing an unprecedented ability to acquire quantitative measurement in a complex heterogeneous cell environment ''in process'' and then to act on those measurements to define highly reproducible methods for cell and colony ''management'' based on application specific critical quality attributes to improve the quality of the manufactured cell lines and cell products.

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Variability in subjective review of umbilical cord blood colony forming unit assay

Cited by 12 publications

References 13 publications

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

Progenitor cells from different zones of human cartilage and their correlation with histopathological osteoarthritis progression

Automated in-process characterization and selection of cell-clones for quality and efficient cell manufacturing

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