Clinical-grade allogeneic amniotic fluid stem cell banking: quality control for therapeutic applications and drug development

Abstract

Background

Allogeneic mesenchymal stem cells (MSCs) offer significant advantages for various medical treatments. However, maintaining an allogeneic MSC bank presents challenges due to concerns about heterogeneity, which directly affects their efficacy. Additionally, issues related to cell senescence can arise even after a short period of serial passaging. Amniotic fluid mesenchymal stem cells (AF-MSCs) demonstrate greater proliferation efficiency compared to other MSCs. They can form clonal cell lines that generate a homogeneous population and can expand in long-term cultures without undergoing cellular senescence. Therefore, this study introduces a method for establishing cell lines and a banking system for AF-MSCs, providing high-quality, authentic human MSCs for clinical applications.

Methods

In this research, we isolated clonal AF-MSCs to select them for the creation of AF-MSC stock through long-term serial passaging. We developed a three-tier cell banking system, which includes an AF-MSC stock, a Master Cell Bank at passage 4, and a Working Cell Bank at passage 9. Standard characteristics were employed to verify identity, safety, and quality control assessments.

Results

Three out of twelve AF-MSC clones were selected to exemplify cell line establishment, the creation of a three-tier banking system, and quality control evaluations to determine their suitability for clinical-grade cell lines in medical applications.

Conclusions

This study offers a comprehensive technical and translational overview of establishing an allogeneic amniotic fluid mesenchymal stem cell bank, tailored for medical applications and drug development.

Introduction

Mesenchymal stem cells (MSCs) have shown great potential for cell therapy and are a crucial component of advanced therapy medicinal products (ATMPs) due to their unique properties. These properties include the ability to self-renew in vitro, differentiate into multiple cell types, reduce inflammation, promote tissue regeneration, have a low risk of immune rejection, and are non-tumorigenic. Moreover, MSCs lack the presence of Major Histocompatibility Complex (MHC) class II, offering an opportunity in allogeneic cell therapy. Numerous research studies and clinical trials have demonstrated the safety and effectiveness of using MSCs from donors to treat various diseases (https://clinicaltrials.gov). However, the therapeutic outcome of stem cell therapy is related to the quality of MSCs. There are controversial results of therapeutic outcomes, most of which are caused by the application of MSC populations lacking homogeneity [1]. To address this issue, it is essential to incorporate MSCs into a stem cell banking system. The system provides stem cells to comply with Good Manufacturing Practices (GMP) and adhere to international regulatory standards set by organizations such as the International Stem Cell Bank Initiative (ISCBI). This involves strict management and ethical evaluation processes, donor selection, tissue derivation, cell cultivation and characterization, cell storage, maintenance of MSC line quality, and quality control measures that comply with regulatory standards for cell therapy products. The banking system helps to ensure the high quality of stem cells and their ready availability for allogeneic therapeutic use.

Most types of MSCs originate from mature or adult tissues that have a limited lifespan after being subjected to repeated subcultures in vitro. A high proportion of the cell population enters a state of senescence, leading to significant changes in cell phenotypes and heterogeneity within the MSC population [2]. These limitations affect the purity and quality of the MSC population, resulting in inconsistent therapeutic outcomes. This issue obstructs the establishment of MSC banking and contributes to a lack of MSC banks that could support allogeneic therapeutic applications and provide raw material for ATMP drug production, so far.

Amniotic fluid-derived MSCs (AF-MSCs) are a type of MSCs sourced from fetal cells found in the amniotic fluid (AF). These cells are established from young cell origins during tissue development during gestation, which provides them with a superior ability to proliferate. AF-MSCs also express several pluripotent markers, including Nanog, OCT4, and SSEA-4. However, unlike pluripotent cells, they do not lead to tumor formation. With highly efficient proliferation capability, AF-MSCs can be easily established into a homogeneous MSC line population from a single cell culture due to their high clonogenicity [3]. Furthermore, their clonal cell line can be subcultured repeatedly throughout 250 population doublings while maintaining their morphology, characteristics, and normal karyotype, retaining long telomeres, and high expression of the tumor suppressor P53 gene [4, 5]. Furthermore, AF-MSCs are a promising stem cell source that has shown significant therapeutic potential in treating various diseases such as osteoarthritis, diabetes mellitus, Alzheimer’s, and Parkinson’s [6]. These cells have unique characteristics, including a low immunogenic profile, Human Leukocyte Antigen-G (HLA-G) presence, immunomodulation with anti-inflammatory effects in response to the environment, multi-lineage differentiation potential, and genomic stability [7, 8]. Given these features, AF-MSCs offer significant benefits by providing a pure and high-quality population of MSCs. Furthermore, establishing a MSC line from a single MSC can be beneficial as it allows for the production of multiple MSC lines from one donor. This approach enhances cost-effectiveness in donor eligibility testing and reduces batch variation when the cells are used in applications. Creating a banking system for adipose-derived MSCs (AF-MSCs) will ensure a continuous supply of MSCs for allogeneic stem cell therapy in the ATMP industry.

This work establishes AF-MSC lines and AF-MSC banking system. The method includes donor screening, cell isolation, establishment and selection of the clonal cell line, creation of initial clone, master, and working cells for the stem cell banking system, and implementation of a quality control procedure. All steps were designed to comply with the regulatory guidelines of the Investigational New Drug (IND) by the Thailand Food and Drug Administration (Thai-FDA) and international standard guidelines [9–13] for the final cell product to support therapeutic applications and ATMPs drug development.

Materials and methods

Ethical approval

Human AF was collected from pregnant women who underwent amniocentesis between 16 and 24 weeks of gestation as part of routine prenatal diagnosis for fetal genetic determination. Prior to participation, each woman received comprehensive information regarding the long-term storage, potential reuse, and possible commercialization of the derived cell lines, in accordance with Institutional Review Board (IRB) guidelines. They were also provided with an individual written consent form. The work was conducted with the approval of the IRB Ethics Committee of Siriraj Hospital (Approval ID: Si 269/2022, Mahidol University, Thailand). The cell lines obtained from these samples will be used for research purposes and distributed under ethical approval.

Donor selection and evaluation

Healthy volunteers who provided AF were carefully selected, following a rigorous process of donor eligibility criteria in compliance with the guideline on ATMPs of Thailand [12] and the international standard of good tissue practice (GTP) guidelines [9, 10]. The donor selection procedure was done via specific donor screening tests, which included general information, a medical history review, a physical examination, and a personal and behavioral assessment questionnaire. Blood analysis was conducted for pathogenic agents via serological and biological testing, as shown in Table 1. Only the AF samples derived from donors who met the guidelines were used for stem cell line establishment.

Table 1.

Donor screening tests for sample collection

Donor screening/testing	Methods	Acceptance criteria
General information	Medical history	Eligible donor
Medical history review	Questionnaire	Accept
Physical examination	Questionnaire	Accept
Personal and behavioral assessment	Assessment	Accept
Blood analysis for pathogenic agents: HBV, HCV, HIV, CMV, HTLV and syphilis	Serology and nucleic acid detection	Negative
Donor record retention	N/A	10 years

Collection and transportation of amniotic fluid samples

AF was collected from pregnant donors under sterile conditions by perinatologists at the maternal-fetal medicine (MFM) unit, Faculty of Medicine Siriraj Hospital, Thailand. A volume of 3 ml of AF was collected into a syringe and immediately kept in a sterile closed box at room temperature (25–30 °C). The sample was delivered to the laboratory within an hour. The parameter for the AF sample after transportation is displayed in Table 2.

Table 2.

Screening parameters of amniotic fluid samples

Processes	Screening/testing	Acceptance criteria
Amniotic fluid collection	Appearance	Clear, pale to straw yellow
	Volume	> 3 ml
	Container	Sterile syringe
Transportation to laboratory	Time: within 4 h	Within 4 h
	Temperature: ambient temperature (25–30 °C)	25–30 °C
	Container: close system	Close system

Isolation and establishment of amniotic fluid stem cell lines

AF-MSC lines were developed from an AF sample following the presence in Phermthai et al. [5]. In brief, the AF sample was centrifuged. The cell pellet was resuspended and cultured in amniotic fluid stem cell culture medium (AFS medium), which contains Minimum Essential Medium α (α-MEM; Gibco, Invitrogen, CA) supplemented with 15% embryonic stem cell-qualified fetal bovine serum (ES-FBS; Sigma-Aldrich, Merck, Darmstadt, Germany), 1% L-glutamine (Gibco), 1% penicillin/streptomycin (Gibco), and 20% Amniomax-II (Gibco) at 37 °C in a humidified environment with 5% CO₂. After that, the medium was changed to remove non-adherent cells. AF cells, characterized by a spindle-fibroblast shape, were harvested using trypsinization and diluted with culture medium before being culture as a single cell in a 96-well plate. Each well containing a single cell was rechecked using an inverted microscope to ensure that a colony was generated from each individual AF cell. Subsequently, these colonies were expanded through 1–3 subcultures to obtain AF clones, which were then cryopreserved for future use. A minimal number of each clone will be further expanded for specific quality testing, including the identification of MSC characteristics according to International Society for Cell and Gene Therapy (ISCT) standards [14] through cell surface proteins using flow cytometry and the ability to differentiate into multiple lineages. Following this, they underwent specific tests for AF-MSC line establishment, including assessment of growth and genetic stability in long-term culture up to subculture passage 15. The AF colony that passed the MSC identification process and growth ability was designated to expand for stem cell banking.

Culturing bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BM-MSCs; ATCC PCS-500-012) were expanded in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% ES-FBS (Sigma-Aldrich), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco). The cells were maintained at 37 °C in 5% humidified CO₂. Media change was performed every 3 days to remove non-adherent cells. The MSCs were grown in a serum-containing medium up to passage 5, after which they were harvested for use in the experiment.

Flow cytometry analysis

Cells have clarified their identity through characteristics of MSC cell surface proteins using flow cytometry. A total of 1 × 10⁶ cells were used per analysis. Cells were harvested and washed in Dulbecco’s Phosphate Buffered Saline (DPBS, Hyclone) and stained with PE-conjugated monoclonal antibodies against CD34 (Thermo Fisher Scientific, #12034942, RRID: AB_1548680), CD45 (Thermo Fisher Scientific, #12045942, RRID: AB_1724079), CD73 (Thermo Fisher Scientific, #12073942, RRID: AB_10733263), CD90 (Thermo Fisher Scientific, #12090942, RRID: AB_10670624), CD105 (Thermo Fisher Scientific, #12105742, RRID: AB_1311123), and HLA-DR (Thermo Fisher Scientific, #12995642, RRID: AB_10698015). Isotype-identical antibodies served as controls for the reaction. Following staining, the cells were washed and fixed with 1% paraformaldehyde and measured with CELL Quest software using a FACSCalibur flow cytometer (Becton Dickinson, NJ).

Differentiation potential analysis

The AF-MSCs were differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro.

For adipogenic differentiation, AF-MSCs were seeded 5 × 10³ cells/cm² and expanded in 10% DMEM. At 80% confluence, the medium was changed to an adipogenic induction medium, which contained DMEM supplemented with 10% Fetal Bovine Serum (FBS; Gibco), 1% penicillin/streptomycin (Gibco), 1 µM dexamethasone (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 5 µg/ml insulin (Sigma-Aldrich), and 60 µM indomethacin (Sigma-Aldrich). AF-MSCs were changed medium twice a week for 21 days. Adipogenic differentiation was detected in intracellular lipid droplet formation using Oil Red O staining (Sigma-Aldrich).

For osteogenic differentiation, AF-MSCs were seeded 5 × 10³ cells/cm² and expanded in 10% DMEM. At 80% confluence, the medium was changed to an osteogenic induction medium, which contained DMEM supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), 0.1 µM dexamethasone (Sigma-Aldrich), 0.2 mM ascorbate-2-phosphate (Sigma-Aldrich), and 10 mM β-glycerophosphate (Sigma-Aldrich). AF-MSCs were medium changed twice a week for 21 days. Osteogenic differentiation was detected in mineralization node formation using Alizarin red staining (Sigma-Aldrich).

For chondrogenic differentiation, AF-MSCs were seeded 5 × 10³ cells/cm² and expanded in 10% DMEM. At 80% confluence, the medium was changed to a chondrogenic induction medium, which contained DMEM supplemented with 1% penicillin/streptomycin (Gibco), 40 µg/ml L-proline (Sigma-Aldrich), 0.1 µM dexamethasone (Sigma-Aldrich), 50 µg/ml ascorbate-2-phosphate (Sigma-Aldrich), 1X insulin-transferrin-selenium (ITS; Gibco), 100 µg/ml sodium pyruvate (Gibco), and 10 ng/ml Transforming Growth Factor beta-1 (TGF-β1; Sigma-Aldrich). AF-MSCs were changed medium twice a week for 21 days. Chondrogenic differentiation was detected in proteoglycan deposits using Alcian blue staining (Sigma-Aldrich).

Evaluation of growth kinetics and genetic stability

The growth kinetics of AF-MSCs were determined by measuring their population doubling time (PDT) and total cell accumulation through extended serial passage [15]. For this, cells of each clone were seeded at a density of 5000 cells/cm² in a 35 mm culture dish (Corning) and incubated at 37 °C in a humidified environment with 5% CO₂ for 3 days. After incubation, the cells were harvested by trypsinization and counted. Cell counting data were collected at every subculture passage to calculate the PDT. PDT was calculated using the following formula: PDT, days = t x log2/(logNH-logNI), where NI is the number of seeded cells, NH is the number of harvested cells, and t is the time of incubation (in days). The cell accumulation was calculated from one cell in the primary culture to passage 15. The PDT should be less than 2 days in every passage.

Establishment of cell bank

Three cryopreservation tiers were employed for MSC banking: AF-MSC stock, a Master Cell Bank (MCB), and a Working Cell Bank (WCB).

The MCB of AF-MSC lines was established using the AF-MSC stock. This AF-MSC stock was scaled up to create a cell line by repeated subculturing and expanded to a maximum of cell passage 4, maintained under conditions of 37 °C in a humidified environment with 5% CO₂. The culture medium was refreshed twice a week to ensure optimal conditions. In-process quality control (IPQC) was conducted at each subculture passage to monitor the quality of the AF-MSC line. Upon reaching passage 4, the cell line underwent quality control specific to MSC lines (MCB-QC). These QC tests were conducted following global medicinal product guidelines [12, 16, 17] as outlined in Table 3.

Table 3.

Quality control tests for establishing amniotic fluid mesenchymal stem cell lines

Attributes	Tests	AF-MSC stock	IPQC	MCB	WCB
General	Cell morphology	•	•	•	•
	Cell viability	•	•	•	•
	Population doubling time (PDT)	•	•	•	•
	Sterility in culture medium 48–72 h	•	•	•	•
	Growth capacity	•
Identity	MSC specific markers	•		•	•
	Adherence to plastic	•		•	•
	Differentiation	•		•	•
Genetic stability	Karyotype analysis			•	•
Purity and safety	Endotoxin			•	•
	Mycoplasma			•	•
	Sterility (fungi and bacteria)			•	•
	Tumorigenicity (in vitro test)			•	•
	HCV/HBV/HIV			•	•
Potency	Immunomodulation			•	•
	Anti-inflammatory action			•	•

After passing the MSC-QC test, the AF-MSC lines at passage 4 that met the criteria were cryopreserved at a concentration of 1 × 10⁶ cells/ml of freezing medium in each cryovial tube (Corning) for further supply as the MCB. The freezing medium comprised α-MEM supplemented with 30% ES-FBS and 7.5% CryoSure-DMSO (WAK-Chemie, Steinbach, Germany). The frozen cells were stored at -80 °C overnight before being transferred to a liquid nitrogen tank.

A WCB of the AF-MSC lines was created from the MCB. The AF-MSCs present in the master cell at passage 4 were expanded to passage 9. During each subculture passage, the cells underwent IPQC testing to ensure the quality of the cell line was maintained throughout the in vitro manipulation process. At passage 9, the cells underwent a quality control assessment (WCB-QC) as detailed in Table 3 before being cryopreserved in 7.5% CryoSure-DMSO at a concentration of 1 × 10⁷ cells/ml of freezing medium. These vials were stored at -80 °C overnight before being transferred to a liquid nitrogen tank for long-term storage.

Randomize quality control for AF-MSC bank

A week after the cell cryopreservation, 1–2 cryopreserved vials from each clone in the MCB and the WCB were randomly selected and thawed to evaluate the quality of the frozen cells according to the quality control tests for MCB and WCB, as detailed in Table 3.

Genomic stability

Karyotype assessments were performed to evaluate the genetic stability of AF-MSCs using the G-banding Technique at an ISO 15,189-accredited laboratory [18]. When the cells reached 60% confluence, they were expanded and harvested for cytogenetic analysis. The cells were incubated with colchicine and then with a hypotonic KCl solution. The supernatant was removed before the cells were fixed with a cold methanol-acetic acid solution (3:1), and the cell suspension was dripped onto a cleaned slide. For G-banding, the slides were treated with trypsin and stained with Giemsa solution. G-banding was evaluated by examining 25 metaphases under a microscope at a magnification of 100X. The analysis detected any numerical and/or structural aberrations [19].

Immunomodulation assessment

The potential of AF-MSCs to modulate the immune system was evaluated by measuring their ability to inhibit the proliferation of T lymphocytes in peripheral mononuclear cells (PBMCs) [20] when co-cultured with AF-MSCs.

AF-MSCs were plated (1 × 10⁴ cells/well) in the upper chamber of a 96-well plate transwell chamber (Corning) and incubated overnight at 37 °C with 5% CO₂. After that, AF-MSCs were treated with 10 µg/ml mitomycin C (MTC; Sigma) to inhibit cell proliferation and incubated at 37 °C with 5% CO₂ for 2 h. Treated AF-MSCs were washed with Roswell Park Memorial Institute medium (RPMI1640; Gibco) 2 times before co-culture with PBMCs.

Heparinized peripheral whole blood was obtained from healthy donors and isolated using a Ficoll-Paque density gradient. The whole blood was diluted 1:1 with DPBS and centrifuged at 800xg for 10 min. The Buffy coat was isolated, diluted 1:4 with DPBS, loaded onto Histopaque-1077 (Sigma-Aldrich), and centrifuged at 400xg for 30 min. The PBMCs were added at ratios of 10:1 (PBMCs: MSCs) into the lower chamber in RPMI1640 supplemented with 10% FBS, and 5 µg/mL of phytohaemagglutinin-L (PHA-L; Sigma-Aldrich) was added to stimulate T lymphocyte proliferation.

The co-cultures were incubated at 37 °C with 5% CO₂ for 72 h. The upper chamber (MSCs) was removed, and the lower chamber (PBMCs) was analyzed for proliferation using Cell Counting Kit-8 (CCK8; Sigma-Aldrich) for 4 h of staining and measuring at 450 nm using a microplate reader Synergy H1 (Biotek, CA).

The index of PBMC proliferation was determined as the ratio of stimulated PBMCs in co-culture and stimulated PBMCs.

Tumorigenicity assessment

The soft agar colony formation assay was used to evaluate the malignant transformation of MSCs by analyzing their anchorage-independent growth [21]. This was done using a CytoSelect™ 96 healthy cell transformation assay kit (Cell Biolabs, Inc., San Diego, CA). The assay was performed according to the manufacturer’s instructions. To begin with, a 1.2% agar solution mixed with DMEM supplement with 20% FBS was added to each well in a 96-well plate and then solidified as a first gel layer. The solid agar in each well was then topped with a mixture of cells (5000 cells /well) and agar, which was prepared by mixing 1.2% agar solution, DMEM supplement with 20% FBS, and AF-MSCs. A positive control was also used, which was the Hela cell. After incubating the plates for 7 days at 37 °C in a 5% CO₂ environment, the agar was solubilized, and the cells were lysed. DNA was stained using CyQuant GR dye before reading the 96-well plate with a multimode microplate reader (Perkin Elmer). To facilitate evaluation, colonies were stained with 0.04% crystal violet for 30 min, washed several times with DPBS, and then detected using a microscope.

Cell senescence assay

Cell senescence was assessed using a Senescence β-Galactosidase staining kit (Cell Signaling Technology, MA), following the manufacturer’s instructions. Briefly, cells were cultured in 6-well plate at an initial density of 5,000 cells/cm² until they reached 50% confluence. The cells were then stained with β-galactosidase and examined using an inverted microscope (OPTIKA IM-3 Inverted Microscope). The percentage of senescent cells was calculated by counting the number of cells stained with intracellular blue dye relative to the total number of cells in 25 random fields at a magnification of 10X.

Cell proliferation assay

Human dermal fibroblast cells (DFs; ATCC PSC-201-012) were co-cultured with MSCs grown in a cell culture insert with 8 μm pores (Corning). DFs were seeded at 5 × 10³ cells/cm² in 6-well plate and cultured in 10% DMEM. MSCs were seeded at 5 × 10³ cells/cm² in the inserts and cultured in 10% DMEM. After 24 h, MSCs containing inserts were added to the DFs wells and flooded with fresh 5% DMEM. Controls consisted of DFs co-cultured with an empty insert. At 48 h, DFs were trypsinized and counted using a hemocytometer.

Anti-inflammation assay

Human monocytic THP-1 monocytes were seeded into a 96-well plate at an initial density of 100,000 cells/well and cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). The THP-1 monocytes were differentiated into M0 macrophages by incubation with 5 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) for 24 h, followed by 72 h of resting in RPMI supplemented with 10% FBS. The M0 macrophages were polarized into M1 macrophages by incubation with 10 µg/ml Lipopolysaccharide (LPS; Sigma-Adrich) and 20 µg/ml Interferon-gamma (IFN-γ; Sigma). Macrophage M2 polarization was achieved by incubating with 20 µg/ml Interleukin (IL) -4 (Sigma) and 20 µg/ml IL-13 (Sigma) for 24 h. Subsequently, M1 macrophages were co-cultured with MSCs (2,500 cells) in the upper chamber of a 96-well plate transwell chamber (Corning) for 24 h. The medium was collected to analyze for the release of IL-1β from macrophages using Lumit^® IL-1β human immunoassay (Promega, WI) according to the manufacturer’s protocols.

Mycoplasma, bacterial and fungi culture test

Microbiological tests were conducted to detect the presence of mycoplasma, bacteria, and fungi in stem cell cultures. The tests were performed by a laboratory service that meets ISO 15189 standards. The detection of bacteria and fungi was assessed through an automated hemoculture. Two methods were utilized for mycoplasma detection: a PCR-based assay and a Mycoplasma detection™ kit (InvivoGen, San Diego, CA). To detect mycoplasma using real-time PCR, the conditioned medium obtained from the AF-MSC culture was collected and centrifuged at 16,000 rpm. The pellet was then diluted with DPBS and used as a sample for the PCR reaction. The reaction was carried out with 2.5 µL of the sample, 2.5 µL of the primer mix, and 10 µL of SYBR green (Roche Diagnostics GmbH, Germany). Amplification was performed under the following conditions: 35 cycles at 95°C for 30 seconds, annealing at 95°C for 1 minute, and extension at 72°C for 1 minute. The expression of β-actin was used as the control housekeeping gene. The mycoplasma-specific primer sequences a forward primer: 5’-ACACCATGGGAGCTGGTAAT-3’ and a reverse primer: 5’-CTTC(A/T)TCGACTT(T/C)CAGACCCAAGGCAT-3’. The specific primer for β-actin consists of a forward primer: 5’-CACGGATCTGAAGGGTGAAA-3’ and a reverse primer: 5’-AGTGGGGTGGCTTTTAGGATG-3’. For mycoplasma detection using the Mycoplasma detection™ kit (InvivoGen), the assay was performed according to the manufacturer’s instructions.

Endotoxin test

The purity of AF-MSCs was evaluated through an endotoxin test using two different methods. The first method involved the kinetic turbidimetric method conducted by a laboratory service that meets the ISO 17,025 standard. The second method, which used the PYROGENT Plus Gel Clot LAL Assays kit (Lonza, Walkersville, MD), had a sensitivity of 0.125 EU/ml and was performed according to the manufacturer’s instructions. Gel clot presence indicates a positive result, while its absence indicates a negative one. The analysis results should be less than 0.2 EU/ml.

Statistical analysis

Data (the mean ± standard deviation; SD) were analyzed using one-way ANOVA with Tukey post-test in PRISM software version 8.0 (GraphPad Software). Statistical significance was set at p-value < 0.05.

Results

AF-MSC line production

On day 7 of the cell isolation process, several clonal cells from AF were observed. The AF clones, which resembled fibroblasts in morphology, displayed a short spindle shape and were relatively small in size (Fig. 1A). We successfully obtained AF clones from 4 out of 5 donors, with each donor yielding more than 15 clones. From these, we randomly selected 23 AF clones from four different donors, with each colony containing approximately 2 × 10⁵ cells. Of the 23 clones, 12 (52%) successfully passed the quality assessment based on their ability to maintain long-term culture capacity for over 15 passages with a low PDT ≤ 2 days. These 12 AF clones included four from donor 1, three from donors 2 and 3, and two from donor 4. They were subsequently evaluated for specific characteristics of MSCs adhering to the ISCT guideline. The results demonstrated their ability to differentiate into adipogenic, chondrogenic, and osteogenic lineages in vitro (Fig. 1B). Additionally, they exhibited positive signals for cell surface markers: CD73 (99.5% ± 0.5), CD90 (87.3% ± 17.7), and CD105 (98.02% ± 2.4), while showing low signals for CD34 (2.9% ± 4.1), CD45 (0.19% ± 0.16) and HLA-DR (0.2% ± 0.14) (Fig. 1C). These clones showed normal karyotypes (46, XX/XY). The 12 selected AF-MSC clones are designated for future stem bank production.

Fig. 1.

Characterization of mesenchymal stem cell (MSC) clones derived from amniotic fluid A Morphological features of AF-MSCs. B The differentiation capabilities of AF-MSCs into adipogenic, chondrogenic, and osteogenic lineages. The scale bar represents 100 μm. C Cell surface markers identified in AF-MSC clones

A three-tiered AF-MSC bank and quality assessment

AF-MSC stock

The 12 selected AF-MSC clones, which successfully passed the MSC characteristic test along with specific tests for growth kinetics and karyotyping, were cryopreserved in liquid nitrogen. This procedure was performed to establish a bank of AF-MSCs at passage 1, in preparation for the MCB.

Master cell bank (MCB)

The AF-MSC clones were thawed and cultured from passage 1 to passage 4 to create MCB. Each subculture passage was evaluated according to IPQC. Three AF clone stocks, SiB1.2, SiB1.3, and SiB2.2, were selected for this presentation. The cells from these 3 cell lines exhibited typical fibroblast morphology (Fig. 2A), maintained a low PDT with an average of 1.20 ± 0.49 days (Fig. 2B), and demonstrated high viability (99–100%) throughout passages 1 to 4. This data can be found in Table 4.

Fig. 2.

Master cell bank and Working cell bank of amniotic fluid-derived MSCs. A Morphology of all 3 AF-MSC lines. The scale bar represents 100 μm. B PDT for each of the 3 AF-MSC lines. C Accumulated cell number for the 3 AF-MSC lines from passage 1 to passage 15

Table 4.

Quality control processes for the production of the AF-MSC lines, along with the corresponding acceptance criteria

Processes	Tests	AF-MSC stock	MCB	WCB	Acceptance criteria
Cell morphology	Microscope observation	Fibroblast	Fibroblast	Fibroblast	Fibroblast
Cell viability (%)	Trypan blue staining	99–100	99–100	99–100	≥ 90
Cell viability after cryopreservation (%)	Trypan blue staining	94.32 ± 4.20	88.73 ± 5.56	91.69 ± 4.59	≥ 80
Population doubling time (PDT)	PDT formula	0.9 ± 0.31	1.20 ± 0.49	1.07 ± 0.24	≤ 2 days
Growth capacity (long term culture)	Microscope observation	15–20 passages	–	–	up to 15 passages
CD73 (%)	Flow cytometry	99.25 ± 1.16	99.54 ± 0.49	98.95 ± 0.09	≥ 90
CD90 (%)	Flow cytometry	98.54 ± 0.65	87.36 ± 17.78	30.85 ± 26.10	≥ 30
CD105 (%)	Flow cytometry	99.20 ± 0.25	98.02 ± 2.42	77.73 ± 31.40	≥ 75
CD34 (%)	Flow cytometry	1.06 ± 0.25	2.90 ± 4.17	2.19 ± 2.82	≤ 5
CD45 (%)	Flow cytometry	0.06 ± 0.06	0.19 ± 0.16	0.08 ± 0.04	≤ 2
HLA-DR (%)	Flow cytometry	0.00 ± 0.00	0.26 ± 0.14	0.16 ± 0.21	≤ 2
Differentiation to adipocyte	Cell differentiation assay	Pass	Pass	Pass	Pass
Differentiation to osteoblast	Cell differentiation assay	Pass	Pass	Pass	Pass
Differentiation to chondrocyte	Cell differentiation assay	Pass	Pass	Pass	Pass
Sterility in culture medium 48–72 h	cell culture media observation	Pass	Pass	Pass	Pass
Endotoxin Level	Kinetic turbidimetric LAL assay	–	< 0.5 EU/ml	< 0.5 EU/ml	< 0.5 EU/ml
Mycoplasma	PCR	–	Negative	Negative	Negative
Sterility (fungi and bacteria)	USP: <71 > Sterility test-direct method	–	No Growth	No Growth	No Growth
Tumorigenicity (in vitro test)	Soft agar colony formation assay	–	Non-tumorigenic	Non-tumorigenic	Non-tumorigenic
HCV/HBV/HIV	PCR	–	Negative	Negative	Negative
Genetic stability	G-banding analysis	–	Normal karyotype	Normal karyotype	Normal karyotype
Immunomodulation	MLR assay	–	–	< 0.50	Proliferation Index < 0.50
Anti-inflammatory; IL-1β level (ng/ml)	Lumit™ Human IL-1β Immunoassay	–	–	0.352 ± 0.00003	As reported

In passage 4, the cells were cryopreserved at a concentration of 1 × 10⁶ cells per vial and stored in liquid nitrogen. The SiB1.2, SiB1.3, and SiB2.2 cell lines successfully passed the quality assessment for MCB-QC and were designated the MCB of AF-MSCs. The results of the quality control assessment for SiB1.2, SiB1.3, and SiB2.2 can be found in Table 4. The total number of cells at passage 4 exceeded 10⁸ cells in each line (Fig. 2C). The MCB from each line is as follows: SiB1.2 has 156 vials, SiB1.3 has 130 vials, and SiB2.2 has 90 vials.

Working cell bank (WCB)

To create a WCB of SiB1.2, SiB1.3, and SiB2.2, a vial from the MCB of each cell line was cultured from passage 4 to passage 9. The cells were evaluated for the IPQC during each subculture passage, as presented in Table 3. All three AF-MSC lines showed homogeneity in fibroblastic spindle shape, maintained a small cell size (Fig. 2A), exhibited low PDT with an average of 1.07 ± 0.24 days (Fig. 2B), and demonstrated 99–100% viability throughout passages 4 to 9. The data can be found in Table 4.

At passage 9, the cells were cryopreserved at a concentration of 1 × 10⁷ cells per vial. To finalize the designation of the cells as the WCB, two cryopreserved vials from each cell line were randomly selected for WCB-QC. All three lines (SiB1.2, SiB1.3, and SiB2.2) successfully passed the WCB-QC quality evaluation and were designated the WCB of AF-MSCs. The total number of cells at passage 9 exceeded 10¹² cells in each clone (Fig. 2C).

Safety of clinical-grade AF-MSC lines

For the safety assessment of the AF-MSC line, we conducted a comprehensive evaluation of safety issues throughout the cell line production process, as detailed in Table 3. We investigated the presence of infectious viral DNA, including HIV, HBV, and HCV, during the donor screening and cell banking stages. Additionally, we assessed various contaminants, including endotoxins, mycoplasma, bacteria, and fungi, at every stage of cell banking. We also evaluated tumorigenicity, which assesses the potential of AF-MSCs to form tumors, during the MCB and WCB, comparing them to BM-MSCs (Fig. 3A-B). Our findings indicated that all AF-MSC lines processed in the stem cell bank were free from contamination by infectious viral DNA and showed no signs of in vitro tumorigenesis.

Fig. 3.

Safety and potency of amniotic fluid- and bone marrow-derived MSCs. For safety, A MSCs were evaluated using a soft agar colony formation assay. HeLa cells served as a positive control, with colony formations indicated by arrows (scale bar: 100 μm). B Quantitative results from the soft agar assay are presented as relative fluorescence units (RFU), comparing AF-MSCs to HeLa cells. For potency, BM-MSCs were used as standard MSCs. C The immunomodulatory capacity was measured by the proliferation index of PBMCs co-cultured with activated PBMCs. D Proliferation potency was shown through the fold change in DFs proliferation when co-cultured with MSCs, with DFs serving as a control. E The anti-inflammatory capacity was determined by measuring IL-1β secreted by M1 macrophages co-cultured with MSCs (n = 4). Results are expressed as mean ± SD, with statistical significance noted by *p-value < 0.05

Potency of amniotic fluid- and bone marrow-derived mesenchymal stem cells

Cell potency of AF-MSCs was evaluated through 3 functional properties, including immunomodulation ability, cell proliferation induction, and anti-inflammation as compared to BM-MSCs. Three cell lines, SiB1.2, SiB1.3, and SiB2.2, using a WCB at passage 9, were used for the analysis.

For evaluation of the immunomodulatory capacity, our analysis focused on lymphocyte proliferation in response to AF-MSCs and BM-MSCs. The results indicated that PHA-induced lymphocytes showed a reduction in the proliferation index of 0.40 when exposed to SiB1.2 (p-value = 0.020), 0.39 with SiB1.3 (p-value = 0.027), and 0.36 with SiB2.2 (p-value = 0.021). This reduction is comparable to a 0.54 decrease in lymphocyte proliferation index observed with BM-MSCs (p-value = 0.064). Our findings suggest that there are no significant differences in immunomodulatory capacity between the WCB of AF-MSCs at passage 9 and BM-MSCs at passage 6 (p-value = 0.147). Moreover, none of the three AF-MSC lines or BM-MSCs induced any lymphocyte proliferation. The index of lymphocyte proliferation is presented in Fig. 3C.

For the effect on cell proliferation, we co-cultured DFs with SiB1.2, SiB1.3, and SiB2.2 using a transwell plate. We observed a significant increase in the proliferation rate of DFs, which showed a 27% increase compared to control cells that were not co-cultured. P-values for SiB1.2, SiB1.3, SiB2.2, and BM-MSCs were 0.001, 0.013, 0.030, and 0.021, respectively. There was no difference in the number of cells between DFs co-cultured with AF-MSCs and those co-cultured with BM-MSCs (p-value = 0.214; Fig. 3D).

For assessing anti-inflammatory action, we focused on the levels of IL-1β secreted by M1 macrophages co-cultured with AF-MSCs. Our results showed that M1 macrophages co-cultured with AF-MSCs secreted significantly lower levels of IL-1β compared to the control non-co-cultured cells, with p-values of 0.037, 0.039, 0.045, and 0.268 for SiB1.2, SiB1.3, SiB2.2, and BM-MSC, respectively. There was no significant difference in the IL-1β levels between those co-cultured with AF-MSCs and those co-cultured with BM-MSCs, with a p-value of 0.186 (Fig. 3E). These findings suggest that AF-MSCs possess anti-inflammatory properties.

Cell proliferation and cell senescence

The proliferation potential of three AF-MSC lines was evaluated by monitoring their growth over multiple subculture passages. Their PDT and total cell counts exhibited a consistent trend through 9 passages (Fig. 2B). Starting from a vial of MCB containing 1 × 10⁶ cells, we can generate approximately 1 × 10¹² cells in WCB at passage 9 (5.7 × 10¹² cells from the SiB1.2 clone, 7.5 × 10¹¹ from the SiB1.3 clone, and 2.2 × 10¹³ from the SiB2.2 clone). By passage 15, the cell counts increase to 1 × 10¹⁸ cells, with 5.8 × 10¹⁸ from the SiB1.2 clone, 9.8 × 10¹⁶ from the SiB1.3 clone, and 7.7 × 10¹⁷ from the SiB2.2 clone. The average PDT was observed to be 1.5 days (Fig. 2B). Moreover, the average PDT between passages 3 and 6 demonstrated that AF-MSCs had a significantly lower PDT compared to BM-MSCs (p-value > 0.0001) (Fig. 4A).

Fig. 4.

Cell senescence observed in amniotic fluid MSCs. A The average PDT of AF-MSCs at passage 3–15 and BM-MSCs at passage 3–6. B The senescence of MSCs was assessed using β-galactosidase staining. The β-gal positive cells are indicated by arrows. The scale bar represents 100 μm. C The percentage of β-galactosidase-positive cells in AF-MSCs compared to BM-MSCs is presented. The results are expressed as mean ± SD, with *p-value < 0.05

For cell senescence, β-galactosidase acts as a biomarker. It appears as blue granules in the cytoplasm following Senescence-associated β-galactosidase (SA-β-gal) staining. The staining results showed that the number of senescent AF-MSCs at passage 9 was low, at only 3.0%. In contrast, the level of cell senescence in BM-MSCs derived from bulk population culture at passage 6 was significantly higher, measuring 39.69% (p-value > 0.0001). These results indicate that AF-MSCs at passage 9 have not undergone excessive senescence and are suitable for use as working cells (Fig. 4B-C).

Discussion

AF-MSCs are promising stem cells that demonstrate significant therapeutic potential for various degenerative diseases. This study outlines the method for deriving an AF-MSC line and conducting quality tests that comply with the standard laboratory practices for Current Good Tissue Practice (CGTP) for human cells and MSC derivation (Fig. 5). The derived MSCs meet the regulatory requirements of starting materials for IND applications as outlined by the Thai-FDA. Additionally, this work describes the processes involved in establishing an allogeneic stem cell bank of AF-MSCs. The strategic approach presented here offers the advantage of creating a large biobank in a cost-effective and resource-efficient manner.

Fig. 5.

Diagram illustrates the production steps and quality control measures throughout the production process of the stem cell bank product

AF-MSCs are stem cells derived from fetal cells during a dynamic period of fetal organ growth and development. These cells demonstrate an intermediate phenotype, existing between pluripotent stem cells and adult MSCs. This unique characteristic endows them with multilineage differentiation potential, a low immunogenic profile, and no tumorigenicity, while also possessing a high proliferation ability similar to that of pluripotent cells. As a result, AF-MSCs demonstrate superior growth and proliferation capabilities compared to other MSC types. Utilizing their unique proliferation properties, we can establish a clonal cell line of AF-MSCs from a single amniotic fluid stem cell using a starter cell method. This technique allows us to generate multiple AF clones from a single donor, with each AF clone serving as an initial AF-MSC stock for developing an AF-MSC line. Furthermore, the derived cell lines demonstrate a homogeneous population, which helps eliminate issues related to mixing different subpopulations that can lead to inconsistent proliferation rates. Phenotypic instability can affect the differentiation potential of each subpopulation, disrupting their predictability in developing specific cell types. Additionally, the heterogeneity within the MSC population may result in varying levels of MHC class II molecules, influencing the immunomodulatory response. Such inconsistencies can pose challenges for quality control and clinical applications [22, 23].

We observed a reduction in the CD90 marker, one of the three surface markers of MSCs as defined by the ISCT, during long-term culture. This finding is consistent with numerous previous reports [24, 25]. CD90 is known to play a role in cell adhesion, migration, differentiation, and signaling. However, our results confirm that the reduced expression of CD90 does not affect the characteristic immunosuppressive properties of AF-MSCs, as demonstrated by the mixed lymphocyte reaction (MLR) assay. This conclusion is supported by the study conducted by Moraes et al. [26], which showed that CD90-negative MSCs can suppress PBMC proliferation similarly to positive MSCs in the MLR assay.

However, there are several important issues that must be considered when creating allogenic MSC banking for therapeutic uses. First, the production of ATMPs is essential to ensure that the cell-starting material complies with GMP. The clinical-grade MSC lines must adhere to these GMP standards, which encompass the entire production process. This begins with rigorous donor screening and continues through cell production and quality testing. Effective governance in this context involves identifying and validating various elements, such as the working area, equipment, chemicals, reagents, personnel, and testing methods. Every aspect of this process must be carefully evaluated to ensure that the produced clinical-grade AF-MSCs are suitable as starting material for ATMPs and therapeutic applications. Secondly, achieving a xeno-free cell culture process is a significant challenge, particularly with AF-MSCs, which struggle to maintain long-term viability in xeno-free media, synthetic supplements, and human platelet lysate. The main obstacle is the difficulty of removing animal-derived materials, as AF-MSCs require high-quality protein sources like embryonic serum-free bovine serum (ES-FBS). Replacing these with synthetic proteins or human platelet lysate can negatively affect cell proliferation and senescence. This challenge likely stems from the fact that AF cells are derived from fetal cells, which share properties with fetal blood serum. The protein composition in fetal serum may be vital for the growth of these cells. If eliminating animal products is not possible, guidelines for human biological medicinal products require stringent viral safety testing for bovine serum. A Certificate of Analysis (COA) must be provided, specifying the serum’s origin and the results of tests conducted for specific viruses. Additionally, it’s also essential to monitor for adventitious agents in cell culture, especially when switching batches of ES-FBS. In our work, we incorporate the evaluation of the immunomodulatory properties of the cells during production and banking at the Master Cell Bank (MCB) and Working Cell Bank (WCB) stages. This helps mitigate the risk of immune reactions and prevent the transmission of animal pathogens. Finally, to ensure compliance with the International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q5D guidelines, a clinical MSC bank must closely monitor cell identity assessments, such as Short Tandem Repeat (STR) analysis. This monitoring should be performed for both the Master Cell Bank (MCB) and the Working Cell Bank (WCB) to minimize the risks of unintentional switching between MSC lines and cross-contamination.

As part of the cell distribution plan, the cryopreserved Cell Product (CP) can be released through one of two approaches: frozen CP or harvested CP. To release frozen CP, the WCB must be transported to the target laboratory using liquid nitrogen or in a suitable container equipped with cooling devices. In the case of harvested CP, the frozen WCB is thawed and cultured at 37 °C until it reaches the expected total cell count. The cells will then be validated through the cell-releasing test (CR-QC) to ensure acceptable morphology, cell viability, sterility, and purity. After passing the validation, the cells should be contained within a closed system of container bottles. They will then be transported in a specifically designed quality container to maintain the required temperature during transit to the target laboratory for further drug processing.

One of the uses of cell products from our AF-MSC bank was in a Phase I clinical trial for osteoarthritis in patients with knee KL scores of 2–3 (Approval ID: Si 233/2565, Mahidol University, Thailand). In this trial, cell products containing 10⁷ cells/vial were released from the bank and transferred to the clinical laboratory within 1 h. After thawing, the cells showed acceptable viability and sterility. This indicates that the processes of cell manipulation and transportation are completely safe.

Conclusion

This work presents an effective process for producing clinical-grade allogenic mesenchymal stem cells (MSCs) derived from amniotic fluid. The goal is to meet the requirements for therapeutic applications and to provide a starting material for the production of ATMPs. We outline the establishment of a three-tier allogeneic MSC banking system to support the production of ATMPs.

Author contributions

TP—conceptualization, methodology, supervision, and writing. ST, SW, PC, JP, TW—methodology and investigation. TW, PA—resources. All authors have read and agreed to the published version of the manuscript.

Funding

Open access funding provided by Mahidol University. This work was supported by a grant No. R016634005 from the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

AF samples were obtained from pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis at the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj hospital, Mahidol University under approval from the IRB Ethics Committee of Siriraj Hospital, Mahidol University, Thailand (Title of the approved project: Stem cell bank for research and medical therapy; approval number: Si 269/2022; date of approval 29 March 2022). All healthy donors have provided written informed consent prior to their participation in the study and the use of their samples. Human DFs were obtained from ATCC (Manassas, VA, with confirmed ethical approval and informed consent from the donors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.