Dataset Information

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

ABSTRACT:

Background

In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources.

Methods

The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated.

Results

Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture.

Conclusions

This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

SUBMITTER: Basatvat S

PROVIDER: S-EPMC10041384 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Publications

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Basatvat Shaghayegh S Bach Frances C FC Barcellona Marcos N MN Binch Abbie L AL Buckley Conor T CT Bueno Brian B Chahine Nadeen O NO Chee Ana A Creemers Laura B LB Dudli Stefan S Fearing Bailey B Ferguson Stephen J SJ Gansau Jennifer J Gantenbein Benjamin B Gawri Rahul R Glaeser Juliane D JD Grad Sibylle S Guerrero Julien J Haglund Lisbet L Hernandez Paula A PA Hoyland Judith A JA Huang Charles C Iatridis James C JC Illien-Junger Svenja S Jing Liufang L Kraus Petra P Laagland Lisanne T LT Lang Gernot G Leung Victor V Li Zhen Z Lufkin Thomas T van Maanen Josette C JC McDonnell Emily E EE Panebianco Chris J CJ Presciutti Steven M SM Rao Sanjna S Richardson Stephen M SM Romereim Sarah S Schmitz Tara C TC Schol Jordy J Setton Lori L Sheyn Dmitriy D Snuggs Joseph W JW Sun Y Y Tan Xiaohong X Tryfonidou Marianna A MA Vo Nam N Wang Dong D Williams Brandon B Williams Rebecca R Yoon S Tim ST Le Maitre Christine L CL

JOR spine 20230110 1

<h4>Background</h4>In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resource ...[more]

PMID: 36994456

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Project description:Abstract Background Repopulating the degenerated intervertebral disc (IVD) with tissue‐specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell‐based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model. Methods Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read‐out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production. Results Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion. Conclusions Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell‐based therapies for IVD regeneration. This study shows that expansion of dog NPCs in higher osmolarity promotes the expression of healthy NPC and progenitor markers at gene and protein level. This population with improved NPC phenotype could have the potential to exert regenerative effects when injected into degenerated IVDs and would therefore be beneficial for cell‐based therapies.

Project description:Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a detergent-free protocol was developed for decellularizing bovine nucleus pulposus (NP) tissues that was effective at removing cellular content while preserving key ECM constituents including collagens, glycosaminoglycans, and the cell-adhesive glycoproteins laminin and fibronectin. Next, novel 2-D coatings were generated using the DNP or commercially-sourced bovine collagen type I (COL) as a non-tissue-specific control. In addition, cryo-milled DNP or COL particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels as a 3-D cell culture platform for exploring the effects of ECM particle composition. Culture studies showed that the 2-D coatings derived from the DNP could support cell attachment and growth, but did not maintain or rescue the phenotype of primary bovine NP cells, which de-differentiated when serially passaged in monolayer culture. Similarly, while bovine NP cells remained highly viable following encapsulation and 14 days of culture within the hydrogel composites, the incorporation of DNP particles within the MCS hydrogels was insufficient to maintain or rescue changes in NP phenotype associated with extended in vitro culture based on gene expression patterns. Overall, DNP produced with our new decellularization protocol was successfully applied to generate both 2-D and 3-D bioscaffolds; however, further studies are required to assess if these platforms can be combined with additional components of the endogenous NP microenvironment to stimulate regeneration or lineage-specific cell differentiation.

Project description:Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.

Project description:BackgroundIntradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.MethodsAfter EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.ResultsTreatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.ConclusionsWJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Dataset Information

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background

Methods

Results

Conclusions

Publications

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

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