An optimized culture system for notochordal cell expansion with retention of phenotype

Background Notochordal (NC) cells display therapeutic potential in treating degeneration of the intervertebral disc. However, research on their phenotype and function is limited by low‐cell yields and a lack of appropriate methodology for cell expansion. Utilizing porcine cells, this study aimed to develop an optimized culture system which allows expansion of NC cell populations with retention of phenotype. Methods Post‐natal porcine and foetal human nucleus pulposus tissue was compared histologically and expression of known NC cell marker genes by porcine NC cells was analyzed. Porcine NC cells were isolated from six‐week post‐natal discs and cultured in vitro under varied conditions: (1) DMEM vs αMEM; (2) laminin‐521, fibronectin, gelatin and uncoated tissue culture‐treated polystyrene (TCP); (3) 2% O2 vs normoxia; (4) αMEM (300 mOsm/L) vs αMEM (400 mOsm/L); (5) surface stiffness of 0.5 and 4 kPa and standard TCP. Adherence, proliferation, morphology and expression of NC cell markers were assessed over a 14‐day culture period. Results Native porcine nucleus pulposus tissue demonstrated similar morphology to human foetal tissue and porcine NC cells expressed known notochordal markers (CD24, KRT8, KRT18, KRT19, and T). Use of αMEM media and laminin‐521‐coated surfaces showed the greatest cell adherence, proliferation and retention of NC cell morphology and phenotype. Proliferation of NC cell populations was further enhanced in hypoxia (2%) and phenotypic retention was improved on 0.5 kPa culture surfaces. Discussion Our model has demonstrated an optimized system in which NC cell populations may be expanded while retaining a notochordal phenotype. Application of this optimized culture system will enable NC cell expansion for detailed phenotypic and functional study, a major advantage over current culture methods described in the literature. Furthermore, the similarities identified between porcine and human NC cells suggest this system will be applicable in human NC cell culture for investigation of their therapeutic potential.


| INTRODUCTION
The increasing incidence of discogenic low-back pain and inability of current treatments to offer long-term resolution has necessitated development of alternative therapies. The drive towards cell-based regenerative therapies for degeneration of the intervertebral disc (DIVD) has resulted in a focus on identification of suitable cell types to drive repair/regeneration. This has included nucleus pulposus (NP) cells, and notochordal (NC) cells, which in humans are only found in the IVD during embryonic/foetal development and throughout childhood. There is compelling evidence to suggest that NC cells are the early progenitors for adult NP cells 1 and that the disappearance of cells with the recognizable NC-like morphology (ie, large, rounded and vacuolated cells) and the onset of DIVD are associated chronologically, which may imply causation. 2,3 As such, NC cells are thought to have substantial therapeutic application and may promote a healthy phenotype in adult NP cells of several species [4][5][6] and NP tissue explants. 7 The NP matrix consists of an irregular mesh of type II collagen and elastin fibers with abundant and highly hydrophilic proteoglycans, most significantly aggrecan. [8][9][10][11][12] DIVD commonly results in a change from type II to type I collagen and a decrease in proteoglycan content leading to dehydration of the NP, 13 an increase in expression of matrix degrading enzymes, [14][15][16][17] an upregulation of inflammatory cytokines [18][19][20] and an increase in cell senescence and apoptosis. [21][22][23] Adult NP cells cultured in NC cell conditioned media (NCCM) showed an upregulation of aggrecan, type II collagen, CD44, link protein and tissue inhibitor of metalloproteinase 1 (TIMP1). [24][25][26][27] In addition, NC cell secreted factors protect NP cells from proinflammatory cytokines, for example, IL-1β, TNF-α, and IL-6, matrix metalloproteinases (MMPs) and activated caspases. 24,[28][29][30][31] These regenerative effects are thought to be mediated via a range of potentially therapeutic bioactive factors including connective tissue growth factor (CTGF). 28,29 Taken together, this evidence provides rationale for the use of NC cells in cell-based regenerative therapies for DIVD and treatment of the associated low-back pain.
However, current culture systems for NC cells, such as the gold standard alginate bead culture, do not allow for proliferation of NC cell populations. 5,6,[32][33][34][35][36] Thus even in commonly used animals where NC cells are retained throughout the majority of lifespan, such as porcine, 7,34,37 canine 24,36 and rat, 38,39 multiple disc levels or multiple animals are required to pool sufficient cells for experimentation. This represents a significant limitation both in current studies investigating NC cell phenotype and function, as well as for future potential clinical translation of human cells.
Such limitations highlight the urgent need for optimized methodologies to expand populations of NC cells without loss of phenotype.
Monolayer culture of NC cells on tissue culture-treated polystyrene (TCP) has been shown to promote proliferation, 32 but has shown poor retention of phenotype, in both porcine and bovine models. 35,40 As such, optimization of culture conditions and consideration of microenvironmental factors are essential to enhance attachment, proliferation and retention of phenotype. Work from Rastogi et al, indicated that choice of media affected NC cell adherence and proliferation, and expression of NC genes such as CD24 in rat NC cells. 38 αMEM and DMEM media were shown to be the most preferable for attachment, and αMEM for retention of gene expression. Coating of two-dimensional (2D) surfaces for monolayer culture has been suggested to promote greater adherence of NC cells, particularly those using laminin isoforms such as 332 and 511. 37,41 In addition to surface coating, substrate stiffness is also an important consideration.
While TCP has a stiffness in the GPa range, the stiffness of a juvenile NP is~0.3 kPa and mature NP~5 kPa 37,42 and soft laminin-coated substrates have been shown to have a profound effect on immature NP cell morphology and gene expression. 43  Here, we used porcine NC cells to develop a system that could be used for expansion of NC cell populations with retention of phenotype over a 14-day culture period. A porcine system was chosen as this system has been shown to contain 80% to 88% NC cells within their NP throughout life and to consistently yield a large number of cells. 37,51 We define a monolayer culture system which allows proliferation of NC cells with significant retention of NC phenotype by consideration of media choice, surface coating, hypoxia, media osmolarity and surface stiffness.

| Porcine sample processing
Porcine carcasses (6-week-old, female, 10-12 kg suckling pigs) were obtained from an abattoir in accordance with local regulations. Spines were removed by dissection under non-sterile operating theater conditions and transferred to a sterile tissue culture hood where individual discs were dissected out. Discs were then either processed individually for histological staining as described above, or NP and AF tissue isolated for gene expression analysis.

| Modification of culture conditions
Culture surfaces were modified though overnight incubation on a shaker at room temperature with 500 μL per well of 2% (v/v) gelatin To test the influence on hypoxia, NC cells were cultured in 2% O 2 , 5% CO 2 and 93% N 2 or 20% O 2 , 5% CO 2 and 75% N 2 for 14 days as appropriate in αMEM media on laminin-521-coated plates. Media was degassed prior to use and all media changes and assays were con- Finally, to assess the influence of substrate stiffness, NC cells were cultured on Softwell Plates containing easy coat gels at 0.5 and 4 kPa or no gel (Cell Guidance Systems, Cambridge, UK), coated with laminin-521 prior to culture with 400 mOsm/L αMEM media in 2% O 2 , 5% CO 2 and 93% N 2 , 37 C.

| Assessment of NC cell viability and morphology
Cells were incubated with 1 mL of 5% Alamarblue in appropriate media at day 3, 7, and 14 timepoints. Plates were incubated at 37 C for 3 hours. Following incubation, 100 μL of 5% Alamarblue in media was removed and read using a BioTek FLx800 at wavelengths 540/35 (ex.) and 590/20 (em.), sensitivity 50.
For lactate dehydrogenase (LDH) assay, media containing nonadherent cells was removed at day three, and adherent NC cells were detached using 1× Trypsin-EDTA for 5 minutes at day three, seven and 14 timepoints. Both populations were lysed using 2% Triton X-100/ HBSS for 1 hour at 37 C in the dark and 100 μL of each solution was transferred to a 96-well plate and combined with 100 μL of reaction mixture (prepared as described in Roche Cytotoxicity Detection Kit).
Plates were incubated for 30 minutes in the dark at room temperature and then read at 490 nm by Thermo Multiskan FC.
At 3, 7, and 14 days, media was removed from live cells and replaced with PBS. These cells were then imaged using an Olympus/ MMI microscope. Estimation of percentage coverage of NC and NPlike morphologies was achieved by eye based on presence/absence of vacuolar structures through division of three low-magnification images into sectors.

| Gene expression analysis
Porcine NP and AF tissue, dissected as described above, was snap frozen in liquid nitrogen and homogenized using a Retsch MM301 tissue homogenizer for 3 minutes, then RNA extracted using TRIzol according to manufacturer's recommendations. RNA was also extracted from cultured NC cells using TRIzol and, in all cases, RNA was reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Fisher Scientific UK Ltd, Loughborough).
Quantitative real-time PCR (qPCR) was performed using the SYBR green method for MRPL19, CD24, KRT8, KRT18, KRT19, and T (Table 1) Figure 5B) and the observed loss of these morphologies occurred in a similar pattern to earlier data ( Figure 4B). Significant differences in expression of selected NC genes were not observed at day 14, although expression of KRT8 was greater and T lower in 2% O 2 compared to 20% O 2 at day seven ( Figure 5C). Hypoxic conditions were considered preferable due to the increase in proliferation and no detriment to phenotype.
No significant differences were observed in cell number ( Figure 6A), retention of NC-like morphologies ( Figure 6B) or expression of selected genes ( Figure 6C)   Alterations to surface stiffness demonstrated greater impacts on proliferation and phenotype (Figure 7). Significantly lower cell numbers were observed on 0.5 kPa surfaces at each timepoint compared to stiffer surfaces. Cultures grown on laminin-coated 4 kPa surfaces showed similar attachment and proliferation to cultures on laminincoated TCP ( Figure 7A). Similar proportions of NC-like morphologies were observed at day seven between cultures on surfaces at 0.5 and 4 kPa and on laminin-coated TCP ( Figure 7B,C). However, cultures on 0.5 kPa surfaces retained a much higher proportion of cells with NClike morphologies (~25%) at day 14, as did 4 kPa surfaces (~20%) compared to the patterns observed in cultures on laminin-coated TCP (both here and in earlier data). It should also be noted that these NClike morphologies were slightly different in appearance based upon the surface stiffness, with morphologies on 0.5 kPa surfaces being more similar to those observed previously described in alginate. There were no significant differences present in the expression of KRT8, KRT18 or KRT19 but a significantly greater expression of T was observed in cultures on 0.5 kPa surfaces ( Figure 7D).

| DISCUSSION
NC cells have been shown to possess a number of therapeutically valuable qualities which make them a potentially ideal cell source for cell-based therapies for DIVD and low-back pain; [4][5][6][7][24][25][26][28][29][30][31]54 however, to date, a method of culture that allows for proliferation while retaining phenotype has not been described. We have developed a NC cell culture system that retains morphology and expression of known NC marker genes in line with the current gold standard, alginate, while also promoting cell proliferation. The first stage in development of this system was to define the suitability of porcine NC as an appropriate model for human NC cells. Known NC markers identified in human NC cells (KRT8, KRT18, KRT19, CD24, and T) 52,53 were found to be expressed in directly extracted porcine NC cells and histological staining demonstrated similarities in morphology and glycoprotein, keratin and GAG content of the extracellular matrix in both human and porcine tissue.
The first difficulty to overcome with design of a NC culture system in monolayer is cell adherence. It has previously been described that NC cells will not adhere to TCP until day six, with only cells of an  If it is assumed that vacuoles exist as a protective measure against hypoosmotic stresses induced by mechanical and loading stresses, 46 it would be reasonable to expect an improvement in morphological retention with hyperosmolar media as had previously been described by Spillekom et al, using a canine model in 3D culture. 36 This loss of morphology was most likely due to the influence of the TCP underlying the surface coating. There is precedent for cells to respond to the stiffness of an underlying culture surface. 67 Therefore, it is likely that the high stiffness of TCP is more prominent than the influence of the surface coating or hyperosmolarity with reference to morphology. This is reinforced by the improved retention of NC morphologies observed at day seven through to day 14 of culture on the more in vivo-like 0.5 and 4 kPa surfaces compared to TCP, highlighting the importance of substrate stiffness in retention of morphology.
It is particularly interesting that while morphology was lost rapidly

| CONCLUSION
Overall, this study defines an optimized system for culturing NC cells, incorporating laminin-521-coated surfaces and αMEM media adjusted to 400 mOsm/L and 2% O 2 . Substrate stiffness is also an essential consideration, with soft substrates promoting retention of phenotype.
Application of such a system will allow for expansion of NC cells with retention of phenotype, hence enabling further phenotypic and functional studies. Adoption of a standardized culture system for expansion of NC cells will enable more robust comparisons between experimental studies from different laboratories to be made. Furthermore, similarities between porcine and human NC cells demonstrated here suggest this system may be appropriate for future transfer to human NC cells to study their regenerative potential in more detail.