UC tissue collection and preprocessing

Umbilical cord (UC) tissue was collected from Shenzhen Baoan District Maternity & Child Healthcare Hospital (Shenzhen, China). The collection process was approved by the Medical Ethics Committee of Shenzhen Baoan District Maternity & Child Healthcare Hospital. The screening, collection, transportation, and infectious disease testing of parturients were carried out according to our standard operating procedures (SOPs). In short, the mothers were between the ages of 20–35 and free from infectious diseases and family genetic diseases and provided written informed consent. After being collected following a cesarean section, the UC (> 20 cm length) was transported to our facility within 24 h at 2–10 °C. Both umbilical cord blood and maternal peripheral blood samples are subjected to pathogen testing, which includes HBV, HCV, HTLV, TP, HIV, EBV, and CMV.

Then, the UCs were weighed, rinsed, decontaminated, and divided into multiple segments. The cord length was estimated and the weight was measured. Subsequently, the cord was rinsed with DPBS (w/o Ca, Mg, Gibco™, USA) and decontaminated using a 0.5% povidone-iodine solution (ADF Hi-Tech Disinfectants, China) for 3 minutes. The cord was then rinsed with DPBS three times to ensure the removal of any remaining blood and disinfectant residue. Using a surgical scalpel, the UC was carefully cut into segments measuring 3–6 cm in length. These UC segments were opened to expose Wharton’s jelly and the underlying blood vessels. Next, two arteries and one vein were carefully removed, and the Wharton’s jelly was extracted. It was then rinsed again before being minced into 1–4 mm3 fragments. These fragments were weighed again and can be further utilized for the isolation of MSCs. The isolation can be performed using either the explant method, where small tissue fragments are directly placed onto the culture medium, or the enzymatic digestion method, which enzymatically breaks down the extracellular matrix to release desired cells for culture. The detailed process is described as follows.

Enzymatic digestion optimization

To optimize the enzymatic digestion method for increased cell yield, various factors need to be taken into consideration, including the selection of enzymes, isolation parameters, and culture parameters. For application in clinical settings, the GMP grade enzyme Collagenase NB6 GMP (Nordmark Biochemicals, Germany) was recommended based on preliminary research conducted on various manufacturer brands. This optimized formulation includes collagenase class I and class II, as well as proteolytic activities such as neutral protease and clostripain. Isolating parameters primarily involve enzyme concentration, digestion time, temperature, and pH, while culture parameters focus on inoculation density after digestion and culture medium. It is worth noting that a temperature of 37 °C and a pH range of 7.0–7.4 have been identified as optimal for enzyme activity. Consequently, a comprehensive assay design was developed, encompassing varying enzyme concentrations (0.2, 0.4, 0.6 PZ U/mL) according to the manufacturer’s instructions, digestion times (2, 3, 4 h), seeding densities (0.5 g, 1 g, 2 g tissue per 75 cm2 flask), and culture mediums (MSC Serum- and Xeno-Free Medium (NutriStem®, Biological Industries, Israel) + 2% hPL (Stemulate®, Sexton Biotechnologies, USA), 5% hPL or 10% hPL). In the experiment, the seeding densities (g tissue per flask) represent the amount of tissue fragments used before digestion and then seeded into a specific size of the culture flask after digestion. We also measured the primary cell densities (cell number after digestion per flask) for analysis. Due to the complex composition of cell components immediately after digestion and significant variations among different samples, we selected post-cultured passage 0 (P0) generation cells as the evaluation indicator for the enzymatic digestion results.

In addition to these factors, the enzymatic digestion method generally follows the previously described basic procedure. Briefly, the tissue fragments were transferred to a bottle and twice more than the volume of collagenase media was added. The collagenase media was prepared by dissolving collagenase powder in DPBS to obtain the corresponding concentration (PZ U/mL). Then, the bottle was placed in a temperature-controlled shaking incubator for digestion, with digestion parameters set to 37 °C and 150 rpm. After digestion, the mixture was neutralized by adding three times the volume of DPBS. Filtration was then performed by passing the mixture through a 100-µm cell strainer (Falcon®, Corning, USA). Subsequently, centrifugation was carried out at 1000 g for 15 min to separate the suspended cells, the supernatant was removed, and the pellet was resuspended in DPBS. Another centrifugation step was performed at 400 g for 10 min to further remove collagenase residue. After discarding the supernatant, the pellet was resuspended in a culture medium. A sample was taken for cell counting (Vi-Cell Blu, Beckman Coulter Inc., USA), and the resulting cell suspension was transferred into a culture flask (Nunc™, ThermoFisher Scientific, USA) for incubation at 37 °C in a humidified atmosphere with 5% CO2. The first medium exchange was performed after 5 days, followed by subsequent medium exchanges every 3 days until days 10–15 or until the cell confluence reached 60-80%. These MSCs were harvested using recombinant trypsin (CTS™ TrypLE™ Select, Gibco™, USA) and designated P0. Cell morphology, quantity, viability, culture time, and immunophenotype were evaluated.

Comparative analysis of the explant and enzymatic digestion methods

A head-to-head comparative study was conducted to analyze the differences between the explant method and the enzymatic digestion method. Briefly, 2 g UC tissue fragments were divided into two equal segments: one was directly added to the culture medium and transferred to 75 cm2 flasks, and the other underwent the collagenase digestion process as described above. WJ-MSCs were harvested at P0 and then seeded for continuous passage at a density of 4500–5500 cells per cm2. Cell morphology, cell count at P0, culture duration, immunophenotype, and population doubling time (PDT) of continuous passage were compared.

Additionally, the seeding density for continuous passage was optimized. Cells at P0 were subcultured at varying seeding densities of 1000 cells/cm2, 3000 cells/cm2, and 5000 cells/cm2 and cultured until P4. Culture time, PDT, and cumulative population doublings (CPDs) were analyzed. The concentrations of glucose and lactate in the culture supernatant were measured daily at P2 to compare metabolism at different seeding densities, using a glucose meter (Roche Diagnostics, Switzerland) and a lactate analyzer (EKA Diagnostics, Germany).

Study of consecutive passaging WJ-MSCs

To assess the manufacturing process and determine the optimal passage number for clinical application, P0 cells obtained using the enzymatic digestion method were consecutively passaged up to P9 at a density of 4500–5500 cells per cm2. The evaluation included the change in cell morphology, PDT, cell viability, and cell diameter with different passages. CPDs were analyzed as well.

Manufacture and scaling up from laboratory scale to pilot scale

All the above experiments were conducted on a laboratory scale based on culture flasks. To further evaluate whether the established method was suitable for GMP-compliant manufacturing, the production process was scaled up to a pilot scale based on a cell factory (Nunc™ EasyFill™ Cell Factory™, ThermoFisher Scientific, USA) and WJ-MSCs were passaged up to P5 (DP). A master cell bank (MCB) and working cell bank (WCB) were established at P1 and P3, respectively. The production and QC of three batches were carried out at the GMP-compliant manufacturing facility operated by Beike Biotechnology (Shenzhen, China).

In detail, first, According to the established isolation protocol, the cells were cultured in a 632 cm2 monolayer cell factory after enzymatic digestion of UC tissue fragments until the cell confluence reached 60-80%. Second, the P0 WJ-MSCs were harvested using recombinant trypsin, washed with resuspension media (DPBS and 5% human serum albumin (HSA) (HuaLan bio, China)) seeded into another monolayer cell factory at a density of 4500–5500 cells/cm2. After 3 days, when cell confluence reached 85 − 95%, the cells were harvested and cryopreserved to establish the master cell bank (P1). Third, the MCB cells were thawed and seeded into a monolayer cell factory at 4500–5500 cells/cm2, resulting in P2 cells. After a 3-day growth period, once cell confluence reached 85-95%, the P2 cells were subsequently harvested and seeded into a 4-layer cell factory to obtain P3 cells, which were cryopreserved to establish the working cell bank. Finally, the WCB cells were thawed and seeded into a monolayer cell factory, resulting in P4 cells at 4500–5500 cells/cm2. After 3 days, when cell confluence reached 85-95%, the P4 cells were then seeded into a 10-layer cell factory to obtain P5 cells, which were cryopreserved for further clinical use. The P1 cells were cryopreserved at a concentration of 5 × 106 /mL, while the P3 and P5 cells were cryopreserved at a concentration of 1 × 107 /mL. Cryopreservation was done using a solution consisting of 7.5% DMSO (Wak-Chemie Medical GmbH, Germany), 20% HSA, and 72.5% multiple electrolytes injection (KeLun Pharmaceutical, China). The cells were filled into AT-Closed Vial® (Aseptic Technologies, Belgium), and immediately frozen using a controlled-rate freezer with a freezing profile of − 1 °C/min. Subsequently, they were stored in the vapor phase of liquid nitrogen at temperatures below − 150℃.

Stability of MCB, WCB, and DP

To validate the stability of pilot-scale production, an extensive series of studies were conducted. Firstly, the MCB, WCB, and DP were subjected to a long-term stability study, whereby they were stored at temperatures below − 150℃ for 0, 3, and 6 months. In addition, multiple freeze-thaw cycles of DP were performed, considering the potential clinical use scenarios. These cryopreserved cells were reanimated by immersing them in a 37 °C water bath. Subsequently, a portion of the cells underwent QC testing. The remaining cells were cryopreserved again as above. This freeze-thaw cycle was repeated three times, with QC testing carried out after each cycle. Furthermore, the in-use stability of DP was assessed. The procedure involved thawing the cells and diluting them with 50mL of 0.9% sodium chloride injection and 10% HSA. The DP was then stored in a drug stability testing chamber with temperatures at 2–8℃ in the dark, or at 20–27℃ with an illuminance of 4500 ± 500 lx. This evaluation was conducted over 0, 2, 4, and 8 h. The QC evaluation encompassed assessing changes in cell viability, cell quantity, expression of cell surface markers, mixed lymphocyte reaction (MLR), and microbial safety testing.

Quality control assaysCell morphology

During the initial day of separating UC-derived MSCs, each culture medium exchange and cell harvest, cell observations were performed using an inverted microscope (IX73, Olympus, Japan). These observations encompassed cell morphology and cell confluence analysis.

Cell counting and viability

The cell numbers and viability were determined by an automatic cell counter (Vi-Cell Blu, Beckman Coulter, Inc., USA) with the trypan blue exclusion method. After mixing Trypan Blue dye with the cell suspension and placing it into the cell counter, the counter captured 100 images for the analysis of cell count, viability, and cell diameter (µm).

Cell proliferation analysis

Cell proliferation analysis included calculating the population doubling time (PDT) and population doubling level (PDL). PDT was calculated by the formula X = T × log2 / (logN - logX0), where T is the time between initial plating and harvest for the respective passage, N is the total number of harvested cells, and X0 is the total number of initial plating [32, 33]. PDL was calculated by the formula X = (logN - logX0) / log2 [34].

Immunophenotype

Surface antigen phenotyping of WJ-MSCs was assessed using flow cytometry (FACSCalibur™, BD Biosciences, USA) and analyzed with CellQuest Pro software. The cells were stained with anti-human antibodies labeled with phycoerythrin (PE), allophycocyanin (APC), fluorescein isothiocyanate (FITC), or PerCP. The specific antibodies used for staining included CD73-PE, CD44-FITC, CD29-PE, CD166-PE, CD45-FITC, CD34-PE, CD14-FITC, CD79a-APC, HLA-DR-PerCP (BD Pharmingen™, BD Biosciences, USA), CD105-APC (eBioscience™, ThermoFisher Scientific, USA), CD90-FITC, CD31-PE, HLA-ABC-APC, CD80-FITC, CD40-PE, and CD86-APC (Biolegend®, BioLegend Inc., USA). Before and following staining, samples were washed with 1× PBS. Isotype antibodies from the same manufacturers for mice or rats were used as controls.

Growth curve and cell cycle assay

WJ-MSCs were initially seeded at a density of 10,000 cells per 12-well plate (Falcon®, Corning, USA). Starting from Day 1 and continuing until Day 7, cells were harvested from three wells every day and counted using an automatic cell counter (Vi-Cell Blu, Beckman Coulter, Inc., USA). The cell counts obtained during the logarithmic growth phase were utilized to calculate the PDT using the listed formula. Further, on Day 3, WJ-MSCs were harvested specifically for the cell cycle assay. The Cycletest™ Plus DNA Kit (BD Biosciences, USA) was employed to determine the cell cycle, and the assay was conducted according to the manufacturer’s instructions. The proliferative index (PI) was calculated by the formula PI= (S + G2/M)/(G0/G1 + S + G2/M) [35].

CFU-F assay

WJ-MSCs were initially seeded at a density of 100 cells per 6-well plate (Falcon®, Corning, USA) in triplicate. After culturing for 10–14 days, the cells were washed with 1× PBS, fixed in 100% methanol for 30 min, stained with 0.1% crystal violet for 45 min, and then rinsed in tap water 2–3 times. Aggregates consisting of 50 cells or more were defined as CFU-Fs (colony-forming units-fibroblasts). The data are reported as the total number of colonies per 100 cells [36–37].

Cellular senescence assay

The cellular senescence assay was performed using a senescence β-galactosidase staining kit (Beyotime Biotechnology, China) following the manufacturer’s instructions. Briefly, WJ-MSCs were seeded in a 6-well plate at a density of 2000–3000 cells and cultured for 72 h. Subsequently, the cells were washed with 1× PBS, fixed at room temperature for 15 min, and then washed thrice with 1× PBS. Following this, the cells were incubated overnight at 37 °C in a dry incubator with the X-gal staining mixture. After washing away the staining solution, the cells were visualized and captured using an inverted microscope (CKX53, Olympus, Japan). Cells displaying a distinct blue staining were recognized as senescent cells. Five images were captured at random fields within each well, with a total of four wells per sample. Representative fields were visualized using a 100× magnification. The number of senescent cells and the total cell count were quantified from the images. The ratio of senescent cells to the total cell count represents the percentage of cellular senescence.

Multilineage differentiation

The differentiation potential of WJ-MSCs was assessed using the OriCell® osteogenic, adipogenic, and chondrogenic differentiation Kit (Cyagen Biosciences Inc., China) according to the manufacturer’s instructions. Briefly, for osteogenic and adipogenic differentiation, the cells were cultured in a 6-well plate at 37 °C with 5% CO2 until reaching approximately 70-100% confluence. Subsequently, the culture medium was replaced with osteogenic or adipogenic differentiation medium and exchanged every 3 days. After 2 to 4 weeks of incubation, the cells were stained with Alizarin Red S solution or Oil Red O solution to evaluate osteogenic or adipogenic differentiation, respectively. For chondrogenic differentiation, (3–4) × 105 cells were used to differentiate into chondrogenic cells for a period of 3 to 4 weeks, which were then stained with Alcian Blue solution.

The stained images were analyzed under an inverted microscope (100 × magnification; CKX53, Olympus, Japan). In each differentiation assay, cells grown in the regular medium were used as the negative control.

Karyotype analysis

Karyotyping was conducted using the Giemsa stain technique. First, cell division was halted in metaphase with 0.3 µg/mL colchicine (Solarbio, China) at 37 °C for 2–3 h. Afterward, the cells were washed and trypsinized, suspended in a warmed hypotonic solution (0.075 M KCl), and incubated for 30–40 min at 37 °C. Following incubation, the cells were washed with a fixative solution consisting of a mixture of methanol and glacial acetic acid at a 3:1 ratio 3 times. Subsequently, the cells were resuspended in a fresh fixative solution and dropped onto clean slides, which were placed in ice water. To obtain G-bands, the slides were dried at a temperature of 70 °C for a minimum of 3 h. Next, the slides were immersed in a 0.01% trypsin solution for 3 min. They were then rinsed twice with saline solution before being stained using a 1:20 dilution of Giemsa solution (Bio Basic Inc., Canada). Evaluation of the band quality was performed under a microscope with a magnification set at 100×. Mitoses were captured using specialized software, and a minimum of 20 metaphases were analyzed in each sample.

Mixed lymphocyte reaction

The mixed lymphocyte reaction (MLR) assay was performed following the protocol described by K. Zhang et al. [38]. In brief, peripheral blood mononuclear cells (PBMCs) were thawed and suspended in PBS and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Solarbio, China). The CFSE-labeled PBMCs were stimulated with 10 ng/ml PHA (Beyotime Biotechnology, China) and cocultured with hWJ-MSCs (5:1) at 37 °C and 5% CO2 in a 12-well plate for 4–5 days. Following the incubation period, all lymphocytes were collected, and cell proliferation was determined by monitoring the gradual reduction in CFSE fluorescence using a BD FACS Calibur flow cytometer. PBMCs without labeling, PHA stimulation, or coculturing were used as negative controls.

Microbial safety test

Microbial safety testing was conducted following the guidelines set third in the Pharmacopoeia of the People’s Republic of China (ChP). The bacterial and fungal tests employed a culture method, while the mycoplasma test utilized both the culture method and the indicated cell culture method. The endotoxin test was performed using the Limulus amebocyte lysate method.

Statistical analysis

Statistical analyses and graphs were performed using GraphPad Prism 8.0. Data are expressed as the mean ± standard deviation (SD). Significant differences between groups were assessed by paired t-tests. A p-value less than 0.05 was deemed to have statistical significance. The correlation between the grams of Wharton’s jelly, the quantities of primary cells after digestion, and the quantities of P0 WJ-MSCs were assessed using linear regression analyses. The coefficient of determination (R2) and p-value were used to evaluate the goodness of fit. A p-value less than 0.05 is considered statistically significant, indicating a strong association between the variables.