From laboratory to clinic: Bridging regulatory and manufacturing gaps in stem cell-based therapies

Abstract

Advanced therapy medicinal products (ATMPs), encompassing therapies derived from mesenchymal stem cells, induced pluripotent stem cells, and their extracellular vesicles, hold substantial transformative potential for clinical applications. However, their translation from bench to bedside and eventual commercialization is hindered by considerable scientific, manufacturing, and regulatory challenges. This review examines these challenges using a structured framework that synthesizes evidence from recent studies, international guidance documents, and regulatory agency reports. We provide an overview of the global regulatory environment and harmonization efforts that influence ATMP oversight, highlighting the frameworks established by major agencies and international initiatives. Technical discussions cover cell sourcing, scalable bioprocess engineering - including bioreactors, closed system technologies, and tangential flow filtration - product quality attributes, and real-time in-process monitoring, incorporating emerging artificial intelligence-driven process analytical technologies. Special focus is given to advanced considerations such as induced pluripotent stem cells tumorigenicity, emphasizing the risks posed by residual undifferentiated cells and the importance of sensitive assays and genetic integrity evaluation; mesenchymal stem cell-derived exosome production, with attention to scale-up strategies, isolation techniques, and quality control standards; global harmonization of good manufacturing practice and ATMP standards; and artificial intelligence-enabled process analytics for automated, closed-cell culture systems. The review further explores potency assays, product comparability, supply chain logistics, and forward-looking trends, including point-of-care manufacturing and digital twin approaches, aiming to equip researchers, developers, and regulators with strategies to advance ATMP development safely and efficiently, ensuring robust therapeutic efficacy and patient safety for next-generation cell and exosome therapies.

Key Words: Advanced therapy medicinal products; Stem cell manufacturing platforms; Global regulatory alignment; Safety and tumorigenicity of induced pluripotent stem cells-derived products; Exosome-based mesenchymal stem cell therapies

Core Tip: This review outlines the major scientific, regulatory, and manufacturing challenges that limit the clinical translation of stem cell-based advanced therapies. It highlights practical strategies - ranging from tumorigenicity assessment for induced pluripotent stem cells and good manufacturing practice-ready production of mesenchymal stem cell-derived exosomes to artificial intelligence-driven process monitoring and digital-twin modeling - that can support safer, scalable, and more consistent development of stem cell and extracellular-vesicle therapeutics.

INTRODUCTION

Stem cell-based and gene therapies, collectively referred to as advanced therapy medicinal products (ATMPs), constitute forefront interventions for a range of debilitating diseases. By harnessing living cells or their derivatives, these therapies offer the potential to regenerate damaged tissues, modulate immune responses, and tackle conditions previously considered untreatable[1,2]. For instance, mesenchymal stem cells (MSCs) mediate their therapeutic activity predominantly through the release of paracrine signals and extracellular vesicles (EVs) that regulate immune functions, while induced pluripotent stem cell (iPSC)-derived cells contribute by directly repairing or replenishing injured tissues[3].

MSCs, for example, are employed for their paracrine immunomodulatory functions and have advanced to late-stage clinical trials targeting graft-vs-host disease and other inflammatory disorders. MSCs mediate their therapeutic effects primarily via paracrine mechanisms, releasing cytokines, growth factors, and EVs that influence immune modulation and tissue repair, including pathways such as the SP1/SK1 axis involved in the regulation of inflammation[1,4,5]. Likewise, iPSCs provide opportunities for personalized or allogeneic cell replacement therapies. In contrast, iPSCs exert their therapeutic potential mainly through directed, lineage-specific differentiation, allowing for the replacement of damaged tissues, while clinical effectiveness relies on precise control of differentiation and the removal of any remaining undifferentiated cells[2,6,7]. Clinical milestones, such as chimeric antigen receptor-modified T cell approvals and MSC-based products in Japan and the European Union (EU), highlight the transformative potential of ATMPs; however, translating these innovations into widespread clinical application presents unique challenges. These therapies combine characteristics of pharmaceuticals, biologics, and medical devices, necessitating large-scale production of living human cells under stringent good manufacturing practice (GMP) conditions, while ensuring sterility, safety, and therapeutic efficacy[1,8,9].

A foundational principle in the manufacturing of ATMPs is that the characteristics of the therapy are fundamentally determined by the production process itself. Even subtle modifications in key operational parameters - such as dissolved CO₂ concentration, hydrodynamic shear forces within bioreactors, or lot-to-lot inconsistencies in poorly defined media components - can drive significant alterations in cellular epigenetic states, phenotypic profiles, and secretome outputs[9,10]. Because each processing step influences subsequent biological behavior, this path-dependent nature introduces inherent biological variability, which contributes substantially to failure rates during clinical translation and large-scale manufacturing. Overcoming inherent biological variability therefore necessitates integrated process engineering, sophisticated analytical strategies, and harmonized quality-control frameworks[10].

Despite their promise, multiple obstacles hinder progress. From a technical perspective, ATMPs must be manufactured at scale with consistent quality while maintaining potency, sterility, and genomic stability. Scientifically, donor-to-donor variability and the absence of well-defined potency markers complicate development and standardization[10,11]. Regulatory bodies worldwide have responded by establishing dedicated frameworks, yet international standards remain fragmented. Harmonization initiatives through International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), Pharmaceutical Inspection Co-operation Scheme (PIC/S), and World Health Organization (WHO) seek to align requirements, but discrepancies persist - for example, differences in facility design between the United States and EU and varying classifications for exosome-based products[12-14].

In this review, we integrate and analyze these challenges using a structured claim-evidence-interpretation framework. We examine the current landscape of manufacturing, including cell expansion, bioreactor technologies, and analytical methods, alongside regulatory considerations such as global guidelines and risk management strategies. Special emphasis is placed on iPSC-associated tumorigenicity, MSC-derived exosome biomanufacturing, standardization efforts, and artificial intelligence (AI)-enabled process control, with the goal of providing a comprehensive roadmap to facilitate the translational success of stem cell therapeutics.

INTERNATIONAL REGULATORY FRAMEWORKS GOVERNING CELL AND GENE THERAPIES

Global regulatory frameworks for ATMPs exhibit considerable variation (Figure 1), presenting significant challenges for developers. International authorities, notably the WHO, stress the importance of harmonizing regulations and promoting reliance among agencies[13,15]. For instance, WHO’s Expert Committee released guidance (TRS 1048 Annex 3) defining classifications for cell and gene therapies, explicitly advocating regulatory harmonization and reliance to minimize barriers to development[8]. The organization also recommends collaborative reviews and shared inspections for cell and tissue products to improve worldwide accessibility[9,16].

Figure 1 Global regulatory landscape for cell- and gene-based therapeutics. A unified comparison of the regulatory systems guiding advanced therapy medicinal product oversight across major regions illustrating regional differences influencing product development and market authorization. EU: European Union; ATMP: Advanced therapy medicinal product; GMP: Good manufacturing practice; FDA: Food and Drug Administration; RMAT: Regenerative medicine advanced therapy; PMDA: Pharmaceuticals and Medical Devices Agency.

Regionally, regulatory standards differ substantially. In Europe, the European Commission has issued ATMP-specific GMP guidelines (EudraLex Vol. 4 Part IV, 2017), modifying conventional GMP practices to address the unique processes of ATMP production, emphasizing risk-based quality controls (QCs)[17]. The EU also maintains a dedicated ATMP regulation (EC 1394/2007), managed by the European Medicines Agency’s Committee for Advanced Therapies. In the United States, the Food and Drug Administration (FDA) regulates cell therapies as biologics through the Center for Biologics Evaluation and Research. Under the 21^st Century Cures Act, the FDA established the regenerative medicine advanced therapy (RMAT) designation, facilitating accelerated approval pathways for qualifying therapies[18]. While the FDA has published guidance on manufacturing and potency assessment, it has not issued separate GMP regulations, instead providing facility design recommendations.

The EU’s “Hospital Exemption”, originally designed to support compassionate, case-specific treatments, presents a regulatory paradox. Although it facilitates timely access at the local level, it also allows the clinical use of products subject to reduced Chemistry, Manufacturing and Controls documentation, leading to variability in regulatory standards among member states[19]. This regulatory divergence can, in turn, discourage sponsors from pursuing centralized marketing authorization and ultimately impede the broader, harmonized rollout of scalable and standardized ATMPs across Europe[20].

The regulatory distinction defined by the “minimal manipulation” criterion remains poorly delineated in practical application, and this lack of clarity has been leveraged by certain direct-to-consumer providers to offer unvalidated therapies under HCT/P exemption pathways[21]. Such ambiguity in oversight not only hampers effective regulatory enforcement but also erodes public confidence and places additional pressure on compliant developers, who must substantiate product quality while navigating multiple - and at times inconsistent-regulatory interpretations[22].

In Asia, Japan’s Pharmaceuticals and Medical Devices Agency (PMDA) enacted the regenerative medicine act (2014), allowing conditional or time-limited approvals for cell therapies, with guidance covering quality evaluation, preclinical testing - including iPSC tumorigenicity assessment - and post-market monitoring[23]. Similarly, China’s National Medical Products Administration has released a series of trial guidelines between 2015 and 2023 addressing stem cell product quality, manufacturing practices, and clinical evaluation, reflecting a rapidly evolving regulatory framework[24]. Even within countries, definitions of products can differ: Exosomes may be regulated as biological drugs in one jurisdiction, yet classified as devices or cosmetic ingredients in another[11,25]. By comparison, the United States FDA’s 21^st Century Cures Act of 2016 introduced the RMAT designation, providing qualifying regenerative products with increased regulatory engagement and pathways that may facilitate expedited approval[26].

Overall, the international regulatory landscape for ATMPs is both comprehensive and fragmented. Although there is growing recognition of the need for ATMP-specific frameworks, inconsistencies in terminology and regulatory approaches - such as “stem cell medicines” in China, “biological ATMP” in the EU, and “cellular biologic” in the United States - require developers to navigate multiple, sometimes conflicting, requirements. This underscores WHO’s emphasis on harmonization and reliance[13,15]. Effective translation of ATMPs demands a clear understanding of each authority’s expectations and the strategic use of harmonization initiatives, such as PIC/S Annex 2A, to align GMP standards internationally[12,27,28]. Table 1 provides a summary of key regulatory guidance and statutes, highlighting the diversity and scope of global requirements.

Table 1 Representative regulatory frameworks for cell and gene therapies (non-exhaustive).

Jurisdiction	Key guidelines/regulations	Remarks
United States (FDA)	RMAT designation (21st Century Cures Act, 2016)[18]; multiple cell and gene therapy guidance documents (e.g., potency testing, viral safety)	Offers expedited development pathways; regulated under biologics framework
EU (EMA)	ATMP GMP guidelines (EudraLex Vol 4 Part IV, 2017)[17]; ATMP Regulation EC 1394/2007	Dedicated GMP annex for ATMPs; emphasizes risk-based quality management
Japan (PMDA)	Regenerative Medicine Act (2014); PMDA technical guidance on cell therapies	Conditional and time-limited approvals; includes nonclinical safety assessments, such as iPSC tumorigenicity[23]
China (NMPA)	Trial guidelines for stem cell products (2015-2023); GMP management updates (2022)[11,19]	Developing regulatory framework with a parallel “drug-like” pathway
WHO	TRS 1048 Annex 3 (2023); previous TRS 878 (1998) tumorigenicity recommendations	Defines ATMP categories and promotes regulatory harmonization[13,15]
PIC/S	GMP Annex 2A for ATMPs (effective May 2021)[12]	Supports global harmonization of GMP requirements, aligned with EU standards
ICH	Q5A(R2) Viral Safety (November 2023)[28]; additional quality modules	Emphasizes viral safety and risk-based management for biotech products

FDA: Food and Drug Administration; RMAT: Regenerative medicine advanced therapy; ATMPs: Advanced therapy medicinal products; EU: European Union; EMA: European Medicines Agency; GMP: Good manufacturing practice; PMDA: Pharmaceuticals and Medical Devices Agency; iPSC: Induced pluripotent stem cell; NMPA: National Medical Products Administration; WHO: World Health Organization; PIC/S: Pharmaceutical Inspection Co-operation Scheme; ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.

Although regulatory frameworks and technological capabilities have advanced rapidly in the United States, EU, and some East Asian countries, global access to ATMPs remains highly uneven. The high costs associated with GMP-compliant facilities, the need for specialized personnel, and the complexity of regulatory submissions create substantial obstacles for low- and middle-income regions, including areas of the Middle East and North Africa. These inequities result in delayed clinical implementation and restricted patient access, highlighting the urgent need for scalable, harmonized, and cost-effective approaches to ATMP manufacturing. Figure 1 illustrates the worldwide regulatory framework for cell- and gene-based therapeutics, emphasizing regional differences that influence the development, evaluation, and market authorization of ATMPs.

BIOPROCESS DESIGN AND MANUFACTURING STRATEGY

Cell source and raw material

The choice of cellular starting material plays a central role in determining the quality of a cell therapy product. Autologous treatments depend on cells obtained directly from the patient, whereas allogeneic products originate from healthy donors or well-characterized cell lines, leading to variability driven by donor age, health, and tissue of origin[1,11,29]. Such heterogeneity is well recognized; for instance, mesenchymal stromal cells derived from bone marrow and umbilical cord differ markedly in their growth behavior and immunomodulatory functions[11]. For example, mesenchymal stromal cells isolated from Wharton’s jelly of the umbilical cord have been shown to display an approximately three- to fourfold higher proliferation rate than bone marrow-derived MSCs, which may reduce the duration required for in vitro expansion[30].

iPSCs provide a more standardized option because an iPSC master cell bank (MCB) can be expanded to generate large, consistent production lots. This advantage was highlighted in a GMP-compliant EV study in which a single human iPSC-derived cardiac progenitor line yielded highly reproducible batches[8,31]. By comparison, earlier MSC-based EV manufacturing often required pooling cells from multiple donors, resulting in considerable lot-to-lot variability[8]. Each sourcing model therefore presents inherent advantages and limitations: Autologous MSCs minimize immune compatibility concerns but introduce high variability and cost; allogeneic systems support scalability but necessitate rigorous donor qualification; and iPSC platforms offer uniformity and expansion potential while posing distinct safety considerations. Accordingly, regulatory guidance mandates comprehensive raw-material testing - including sterility, identity, and viability assessments for each newly introduced donor or cell line[1,8,32] - and best manufacturing practice recommends banking cells at low passage and using serum-free, xeno-free GMP-grade media to reduce risks of contamination and genomic instability[1,8,33].

In addition to MSCs and iPSCs, novel strategies are exploring the use of endothelial cells, tissue-specific progenitor populations, or reprogrammed somatic cells, either individually or in synergistic combinations, to boost functional integration and therapeutic outcomes. These approaches have the potential to enhance vascularization, target tissue specificity, and engraftment efficiency, though they also increase both manufacturing challenges and regulatory considerations[34,35]. Figure 2 presents a schematic overview of cell sourcing strategies for ATMPs, comparing autologous, allogeneic, and iPSC-based approaches. It highlights differences in donor variability, immune compatibility, scalability, and reproducibility, while emphasizing critical quality and safety measures such as sterile cell banking and the use of GMP-compliant culture media.

Figure 2 Diagrammatic representation of cell sourcing approaches for advanced therapy medicinal products. The schematic contrasts autologous, allogeneic, and induced pluripotent stem cell methodologies, emphasizing distinctions in donor heterogeneity, immune compatibility, scalability, and reproducibility of production. Key elements, including sterile cell banking and the application of good manufacturing practice-compliant culture media, are illustrated as critical measures for maintaining cell quality and safety. GMP: Good manufacturing practice.

Upstream bioprocessing: Cell culture and expansion

Clinical-scale cell expansion requires sophisticated bioprocessing strategies that go well beyond traditional T-flasks and two-dimensional (2D) culture systems, which cannot satisfy the demands of therapeutic applications. Advanced bioreactors - such as stirred-tank, perfusion, and 3D microcarrier platforms - offer enhanced surface area or volumetric capacity, supporting the controlled proliferation of cells, including mesenchymal stromal cells, as exemplified by the Xuri Cell Expansion system[8,11,36]. Automated, closed-system devices, like Miltenyi Biotech’s CliniMACS Prodigy, seamlessly integrate cell cultivation with automated feeding and harvesting, while critical process parameters - including pH, oxygen tension, and nutrient availability - are continuously monitored and managed through fed-batch or perfusion strategies to maximize yield. Under GMP-compliant conditions, all components, including media and reagents, are upgraded to GMP-grade, frequently using serum-free formulations, to meet regulatory and safety standards[8,37,38]. This approach parallels biopharmaceutical production by creating tightly controlled environments that minimize contamination risks and enhance batch reproducibility. Nonetheless, scaling introduces specific challenges: Shear forces in stirred systems can impair cell viability, and continuous perfusion demands careful regulation to maintain optimal nutrient levels. Each expansion platform must be rigorously qualified under GMP to confirm cell growth, viability, and functional potency. Consistency remains paramount, with studies demonstrating that initiating production from a well-characterized iPSC MCB yields more reproducible results than primary MSCs sourced from multiple donors[8,39,40]. To optimize outcomes, developers strategically balance batch vs continuous operations and frequently employ 3D or microcarrier cultures to enhance cell density and overall process efficiency. For instance, a study employing a fed-batch vertical-wheel bioreactor reported that human mesenchymal stromal cell densities of approximately 2 × 10⁵ cells/mL to 6 × 10⁵ cells/mL were reached within five days, demonstrating the substantial productivity attainable with well-optimized bioreactor systems[41].

McKee et al[36] engineered a self-assembling hydrogel system composed of polyethylene glycol and dextran to support embryonic stem cell growth. Using this 3D matrix, they demonstrated that embryonic stem cells could be reliably expanded on a weekly basis while maintaining cell viability at levels similar to those achieved in standard 2D cultures[36]. In stirred-tank bioreactors, hydrodynamic shear represents the dominant critical process parameter, and its impact is more accurately described using variables such as the maximum energy dissipation rate (ϵ_ax) and wall shear stress (τw), rather than impeller speed alone[42]. When ϵ_ax is too high, cells may undergo apoptosis or experience shifts in mechanotransduction signaling pathways (including RhoA/ROCK and YAP/TAZ), leading to altered differentiation tendencies or modifications in their secretory profiles. Conversely, insufficient agitation can impair mass transfer, resulting in hypoxic zones within the culture[8]. Establishing a robust process design space therefore requires integrating hydrodynamic simulations, real-time sensor analytics, and cell-level functional assessments to optimize mixing while minimizing mechanosensitive cellular injury[43,44].

In high-density perfusion systems, cells frequently exhibit enhanced aerobic glycolysis (the Warburg effect), leading to rapid lactate accumulation and elevated pCO₂ levels that can compromise both growth and functional performance[45]. Strategies to mitigate these effects include implementing high-rate perfusion using cell-retention technologies - such as tangential flow filtration (TFF) or alternating tangential flow modules - combined with optimized, chemically defined, xeno-free media to manage metabolite accumulation while maintaining cells for continued proliferation[46,47].

Table 2[48-50] provides an overview of indicative cell expansion outputs documented for widely applied culture systems, including two-dimensional multilayer platforms (such as CellSTACK and Hyperflask), stirred-tank bioreactors configured with microcarriers or vertical-wheel mechanisms, and hollow-fiber bioreactors characterized by high surface-area cartridges. The table outlines the approximate cell densities (cells/mL) attainable with each approach and highlights that these values are influenced by factors such as the cellular origin, starting inoculum density, media composition, and downstream harvesting or resuspension conditions.

Table 2 Representative cell expansion yields across culture platforms[48-50].

Culture platform	Typical cell density (cells/mL)
2D Multilayer Systems (CellSTACK, Hyperflask)	Approximately 2 × 105 to 6 × 105
Stirred-Tank Bioreactors (microcarrier/vertical-wheel)	Approximately 5 × 105 to 1.7 × 106
Hollow-Fiber Bioreactors (high surface area cartridges)	Approximately 1 × 107 to > 1 × 108

Reported yields vary depending on cell source, seeding density, media composition, and harvest/resuspension volume.

Figure 3 presents a conceptual schematic of a scalable cell production strategy utilizing the CliniMACS Prodigy platform, demonstrating microcarrier-supported cell expansion within a bioreactor configured as a closed, automated system. The illustration underscores continuous surveillance and regulation of key culture parameters - such as pH, dissolved oxygen tension, and nutrient concentrations - to maintain controlled growth conditions, enhance process consistency, and support reproducible cell manufacturing aligned with translational and regulatory standards.

Figure 3 A diagrammatic representation of a scalable cell culture process utilizing the CliniMACS Prodigy platform is presented. In this workflow, cells are cultured on microcarriers inside a bioreactor, subsequently undergoing growth within an automated, closed system. Critical environmental factors such as pH, dissolved oxygen, and nutrient levels are continuously monitored and regulated to maintain ideal conditions for consistent cell proliferation and reproducibility.

Downstream processing: Cell recovery and product purification

Downstream processing and the associated cost of goods remain key limiting factors in ATMP manufacturing. Employing closed, automated separation platforms - such as continuous-flow centrifuges (e.g., Sepax or kSep) and TFF circuits - minimizes contamination risks and reduces operator-dependent variability while allowing simultaneous cell concentration, washing, and formulation[45]. Additionally, thermoresponsive surfaces (such as pNIPAAm) and other non-enzymatic harvesting methods help maintain essential surface markers, and rigorously optimized cryopreservation protocols - including DMSO alternatives and validated cooling profiles - are critical for defining product shelf-life and enabling reliable global distribution[48-50].

Downstream processing of cell and exosome therapeutics necessitates methodologies that are simultaneously gentle, to preserve product integrity, and robust enough to meet clinical-grade purity standards. For cell therapies, this involves detaching adherent cells through enzymatic or non-enzymatic approaches, collecting them via centrifugation, washing to remove residual reagents, and concentrating the product for either cryopreservation or direct infusion[11,51]. EV-based products require additional purification due to their small size and complexity; TFF, for example, is frequently employed to process large volumes of conditioned media and enrich EVs[52]. Other commonly used methods, including ultracentrifugation, size-exclusion chromatography, and affinity capture, each present specific trade-offs: While ultracentrifugation is widely regarded as the “gold standard”, it is labor-intensive and may co-purify contaminants, whereas size-exclusion chromatography and immunoaffinity techniques can enhance purity but may reduce yield[11]. Downstream workflows must carefully control shear forces and thermal exposure to safeguard cellular or vesicular integrity, while complying with GMP standards that require single-use sterile systems, closed processing environments, and fully validated procedures. Exemplifying this, a GMP-compliant study demonstrated an end-to-end workflow from closed-system vesiculation to sterile filtration into final product vials[8]. Such validated processes are critical for regulatory submissions, including Investigational New Drug applications, to demonstrate product safety, stability, and reproducibility. However, exosome therapeutics face additional challenges, including the lack of standardized potency assays and heterogeneous isolation protocols, which remain major bottlenecks; overcoming these limitations will necessitate the establishment of new regulatory and methodological guidelines[11,52]. Figure 4 illustrates the workflow for producing cell- and exosome-based therapeutic formulations. The process begins with cell detachment, followed by centrifugation to separate cells from the culture medium. Subsequent purification removes contaminants and enriches the desired vesicles or cells, e.g., via TFF or chromatography and the final sterile filtration step ensures microbiological safety and consistent product quality suitable for therapeutic applications.

Figure 4 Overview of cell and exosome therapeutic preparation pipeline. This illustration outlines the stepwise procedure used to produce exosome- and cell-derived therapeutic formulations. The workflow initiates with cell detachment, after which collected material undergoes centrifugation to separate cellular components from the culture medium. The product is then subjected to purification, enabling removal of unwanted contaminants and enrichment of target vesicles or cells. The final stage, sterile filtration, ensures microbiological safety and product consistency, resulting in a preparation that meets standards for therapeutic use. QC: Quality control.

QC and analytics

Comprehensive QC is essential to guarantee the identity, purity, potency, and safety of ATMPs. Regulatory authorities require extensive in-process and release testing, which for cell therapies typically encompasses sterility assessments to prevent microbial contamination, endotoxin and mycoplasma testing, viability measurements, immunophenotyping for characteristic markers, and functional potency evaluations. Guidance from the European Pharmacopeia and international bodies, such as the International Society for Cell & Gene Therapy marker panels for MSCs, provides a framework for these analyses[53-55]. In the context of EV production, QC strategies follow Minimal Information for Studies of EVs 2018 and European Pharmacopeia standards, including particle sizing via nanoparticle tracking analysis, surface marker profiling with MACSPlex, and functional bioassays[8]. Additional essential evaluations comprise genetic stability assessments through karyotyping or comparative genomic hybridization and viral safety testing aligned with ICH Q5A and Q5A(R2) guidelines. Achieving reproducible manufacturing demands consistent expression of critical markers and maintenance of functional properties, such as differentiation capacity or immunomodulatory activity[1]. Potency assays remain complex due to multifaceted mechanisms of action, and regulatory bodies may accept well-justified surrogate assays when supported by comparability data. QC practices continue to advance, with updates like ICH Q5A(R2) placing greater emphasis on viral clearance and the adoption of novel assays, including polymerase chain reaction-based detection of residual plasmid or vector DNA. Furthermore, all raw materials, including culture media and growth factors, should be GMP-grade or thoroughly tested to support a risk-based quality strategy[8]. Collectively, a rigorous QC framework underpins the safety, efficacy, and regulatory compliance of both cell- and exosome-based therapeutics.

For pluripotent stem cell platforms, regulatory expectations mandate a multi-tiered QC framework, including comprehensive characterization of the MCB, routine molecular cytogenetic assessments (such as karyotyping and array-comparative genomic hybridization), and next-generation sequencing-based monitoring to detect sub-clonal genetic alterations[56]. While cGMP-compliant iPSC banks with thoroughly documented characterization protocols are increasingly available for preclinical and clinical applications, sponsors are required to demonstrate consistent genetic stability across both MCBs and working cell banks to meet regulatory safety standards throughout the product lifecycle[57,58].

Automation process control and digital analytics

The integration of automation, real-time process monitoring, and AI-driven analytics presents a transformative opportunity to improve the reliability and efficiency of ATMP manufacturing. Conventional manual cell culture methods are prone to human error and contamination, whereas automated platforms - including liquid-handling robots and closed bioreactors - reduce variability and support continuous operation. As noted in recent literature, the implementation of “automatic, closed system(s) that allow real-time monitoring of QC” is recommended to mitigate errors and contamination risks[1,59]. Process analytical technology (PAT), incorporating in-line sensors and software, has been increasingly applied in cell and gene therapy production, with techniques such as online Raman spectroscopy and dielectric spectroscopy used to monitor cell density, nutrient consumption, and metabolite accumulation in real time[60-62]. Emerging sensor technologies, such as the 908 Devices platform, enable continuous measurement of glucose and lactate, providing real-time data to AI algorithms that predict culture outcomes. This strategy aligns with Pharma 4.0 principles, representing the digitization of biomanufacturing processes. By applying AI and machine learning (ML) to extensive datasets - including temporal, imaging, and sensor data - manufacturers can anticipate cell growth patterns, detect process deviations early, and enhance batch-to-batch consistency. Although AI applications in cell and gene therapy remain limited, experience from other biologics indicates that deep-learning models trained on cell morphology imaging can non-invasively assess cell health[1,60,63]. Regulatory agencies are beginning to address AI integration, exemplified by the European Medicines Agency (EMA)’s 2024 report recognizing AI/ML utility in manufacturing and quality oversight[60]. Effective implementation requires comprehensive data infrastructure and thorough algorithm validation. Currently, automated closed systems with integrated digital monitoring represent best practice for ATMP production[1,60]. Overall, AI-enabled analytics have the potential to lower manufacturing costs and reduce failure rates in cell therapy processes, though further technological development and regulatory guidance are essential.

AI- and ML-enhanced PAT, including Raman and dielectric spectroscopy, imaging modalities, and deep-learning-based morphology classifiers, hold significant potential for non-invasive, near-real-time monitoring of cellular states[64]. Nevertheless, most applications remain at the proof-of-concept stage and require comprehensive algorithm validation, robust data management systems, and detailed regulatory lifecycle documentation before integration into GMP-compliant workflows. Recent reviews highlight best practices for AI-driven quality monitoring, underscoring the importance of well-curated training datasets and model interpretability to facilitate regulatory acceptance[59]. Table 3 summarizes the key analytical and QC assays commonly applied to ATMP manufacturing, recognizing that the list is illustrative rather than exhaustive. Detailed potency testing strategies and stability study designs are developed individually for each product in accordance with ICH and major pharmacopoeial standards, including guidance such as ICH Q6B and Q1A. Figure 5 depicts an advanced bioprocessing framework that integrates automated production systems with continuous online monitoring using Raman and dielectric spectroscopy. Real-time data are analyzed through AI-driven models, providing dynamic insights that enhance process control, reliability, and overall robustness in biomanufacturing workflows.

Figure 5 Smart bioprocessing framework integrating automation, inline sensing, and artificial intelligence-enabled interpretation. This illustration depicts the use of automated production systems coupled with continuous analytical monitoring through online Raman and dielectric spectroscopy. These tools generate real-time process data that feed into artificial intelligence-based analytical models, enabling dynamic insights, improved process control, and greater robustness across biomanufacturing workflows. AI: Artificial intelligence.

Table 3 Critical quality attributes and assays for advanced therapy medicinal product manufacturing.

Attribute	Example assays/methods	Guideline references
Identity	Flow cytometry (cell surface markers), PCR for unique genes	EMA/ICH guidance, pharmacopoeias
Purity	Sterility testing, mycoplasma PCR, endotoxin (LAL assay)	Ph.Eur./USP
Viability	Dye exclusion (trypan blue, 7-AAD), metabolic activity assays	CBER/EMA guidance
Potency	Functional bioassays (e.g., MSC-mediated T-cell suppression)	Case-specific; often ICH Q6B
Genetic stability	Karyotype, array CGH, NGS for mutations	CBER draft guidance, ISSCR recommendations
Tumorigenicity	Soft agar colony formation, injection into immunodeficient mice[23]	WHO TRS 878, FDA draft guidance
Viral safety	Filtration of spent media, adventitious virus testing panels	ICH Q5A(R2)[27]
Exosome-specific	Particle size analysis (NTA), exosome marker proteins (CD63, CD81)[16]	MISEV2018 standard

PCR: Polymerase chain reaction; EMA: European Medicines Agency; ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use; Ph.Eur.: European Pharmacopoeia; USP: United States Pharmacopeia; 7-AAD: 7-aminoactinomycin D; CBER: Center for Biologics Evaluation and Research; MSC: Mesenchymal stem cell; CGH: Comparative genomic hybridization; NGS: Next-generation sequencing; ISSCR: International Society for Stem Cell Research; WHO: World Health Organization; FDA: Food and Drug Administration; NTA: Nanoparticle tracking analysis; MISEV2018: Minimal Information for Studies of Extracellular Vesicles 2018.

SAFETY CONSIDERATIONS: TUMORIGENIC POTENTIAL AND CELL-RELATED RISKS

Therapies derived from iPSCs

iPSC-derived therapeutic products entail an intrinsic risk of tumor development if undifferentiated or improperly reprogrammed cells remain within the administered preparation. Owing to their full pluripotency, iPSCs can generate teratomas, and even well-designed differentiation protocols may leave behind small fractions of undifferentiated or only partially matured cells with tumorigenic potential[6,15,65]. This risk is heightened when reprogramming methods involve oncogenic drivers such as c-Myc or utilize viral vectors that may introduce genetic instability. As highlighted in the Oxford review, differentiated iPSC cultures are inherently heterogeneous, underscoring the need for meticulous screening to eliminate malignant or pre-malignant impurities[15,66]. Consequently, regulatory agencies require comprehensive tumorigenicity assessments. The Japanese PMDA, for instance, mandates separate evaluation of both the iPSC master bank and the final therapeutic construct[23], recommending a suite of in vitro assays - such as soft agar and focus formation tests - as well as in vivo studies using immunodeficient mouse models, consistent with WHO TRS 878 guidance[23]. Thorough genomic assessment is equally critical, as chromosomal anomalies, copy-number variations, and epigenetic irregularities are well-recognized drivers of tumor risk in iPSC-derived cells[6,67,68].

The growing clinical use of gene-edited cellular therapies has heightened regulatory focus on CRISPR-Cas technologies, especially concerning unintended mutations, chromosomal alterations, and potential long-term genotoxic effects. Contemporary guidelines recommend thorough assessment of off-target modifications through advanced sequencing methods, longitudinal tracking of edited cell clones, and incorporation of genome stability data into safety evaluations guided by risk-based frameworks.

Overall, current evidence demonstrates that iPSC-based therapies demand layered and stringent safety controls. Regulators expect a combination of orthogonal testing approaches - including culture-based assays, genome-level analyses, and animal models - to thoroughly identify and eliminate cells with tumorigenic potential[23,69]. In practice, developers employ strategies such as marker-based cell sorting, suicide-gene safeguards, and extensive genomic quality assurance. PMDA guidance further emphasizes that even the foundational iPSC “stock” must exhibit minimal susceptibility to malignant transformation[23,70]. Because very small numbers of residual pluripotent cells - estimated at roughly 10³-10⁴ - could be sufficient to initiate neoplastic growth in vivo[23], robust screening methods and reliance on well-characterized, low-passage cell banks are essential. Demonstrating genomic stability through karyotypic and mutational analyses[6,71], together with evidence of absent teratoma formation in animal studies, remains a central requirement for regulatory approval. Collectively, these principles support a preventive strategy: Address potential risks at the earliest stage of development and validate safety thoroughly throughout product manufacturing and evaluation[6,23].

MSCs and other adult-derived cellular therapies

MSC therapies are generally associated with a lower intrinsic risk of tumor formation compared with iPSC-based products, yet safety considerations remain essential. MSCs are adult somatic cells with limited proliferative potential, which has led to their characterization as “minimally tumorigenic”. Clinical experience, including trials with products such as Alofisel and TEMCELL, has not documented MSC-induced neoplasms. Nonetheless, extended in vitro expansion of MSCs can lead to chromosomal alterations[1,72,73], and isolated instances of malignant transformation have been observed in rodent models. Regulatory guidance, including EMA’s Committee for Advanced Therapies reflection papers, emphasizes that all cell lines used for biologics production must originate from non-tumorigenic sources[28,42]. Professional organizations such as the International Society for Stem Cell Research and International Society for Cell & Gene Therapy advise ongoing karyotype surveillance and screening for oncogenic viruses, despite the immunomodulatory characteristics of MSCs.

Taken together, these observations indicate that MSC therapies are largely safe, but rigorous adherence to GMP culture practices is required to maintain genomic stability and sterility. While the overall claim is that MSCs carry a lower tumorigenic risk than iPSCs, they are not entirely free of risk. Evidence supports the implementation of routine assessments for cellular transformation, including long-term culture monitoring and soft agar assays. Clinical products like remestemcel-L have progressed to late-stage trials with favorable safety profiles, demonstrating that these risks can be effectively managed. Nevertheless, developers must comply with regulatory expectations: Allogeneic MSC banks should be carefully screened to confirm the absence of tumorigenic viruses and chromosomal abnormalities across both master and working cell banks. Ultimately, maintaining high standards of product purity, identity, and genetic stability remains critical, even for cell types considered relatively safe.

Figure 6 illustrates the differences in safety and manufacturing requirements between iPSC-derived and MSC-based therapies. iPSC-derived products may harbor residual undifferentiated cells, genomic abnormalities, or heterogeneous differentiation, increasing their tumorigenic potential. In contrast, MSCs present a lower risk of malignant transformation and benefit from well-established QC frameworks, including routine genomic monitoring and adherence to GMP standards, supporting safer and more predictable therapeutic applications.

Figure 6 Comparison of safety risks and manufacturing requirements for induced pluripotent stem cell-derived products vs mesenchymal stem cell-based therapies. This figure highlights fundamental differences in biosafety profiles between induced pluripotent stem cell derivatives and mesenchymal stem cells. Induced pluripotent stem cell-derived cell populations may retain undifferentiated cells, accumulate genomic abnormalities, or exhibit heterogeneous differentiation states, all of which heighten tumorigenic risk. In contrast, mesenchymal stem cells demonstrate a substantially lower propensity for malignant transformation and benefit from established quality frameworks, including routine genomic monitoring and compliance with good manufacturing practice, supporting their more predictable and safer use in therapeutic production. iPSC: Induced pluripotent stem cell; MSC: Mesenchymal stem cell.

CUTTING-EDGE DEVELOPMENTS AND NOVEL DIRECTIONS

Therapeutic applications of MSC-derived EVs

Exosomes produced by MSCs (MSC-EVs) offer a compelling cell-free therapeutic approach, delivering many of the beneficial effects of MSCs - such as immunomodulation - without the risks associated with engrafting proliferative cells[4,11]. Early-phase clinical investigations are exploring MSC-EVs for conditions including ischemic injury and inflammatory diseases[74].

A notable advantage of this strategy is its scalability: MSCs can be expanded in culture, their exosomes collected, and the parental cells removed, thereby reducing tumorigenic risk. Nonetheless, clinical translation faces significant challenges related to manufacturing and regulatory oversight. As noted by Zhang et al[11], producing exosomes at scale necessitates cGMP-compliant bioreactor systems and rigorous quality management, yet their regulatory classification differs across regions, with exosomes potentially treated as biologics, drugs, or medical devices. Furthermore, standardization of potency assays remains unresolved; current QC methods often rely on markers like CD63, which may not accurately represent functional activity[11].

International Society for Extracellular Vesicles and recent literature highlight EV heterogeneity as a key obstacle for clinical translation, with the Minimal Information for Studies of EVs guidelines serving as the community standard for characterization[17,75]. In practice, developers have mitigated variability by employing single-donor or clonal parental cell sources, closed vesiculation systems, and scalable TFF-based workflows to generate GMP-compliant EVs, strategies that also help navigate current regulatory ambiguities regarding EV classification and potency assessment[76].

Collectively, these factors indicate that while MSC-EVs avoid some of the risks inherent to cell-based therapies, they introduce unique hurdles. Regulatory uncertainty and variability in isolation and characterization methods present challenges for developers. Effective approaches have been demonstrated, such as the use of human iPSC-derived cardiac cells processed in a closed vesiculation system with TFF under full GMP conditions[8]. Key elements of such strategies include using single-donor or clonal sources to minimize variability, scalable TFF for concentration, and validated sterility measures. Until comprehensive international standards for EV therapies are established, each product must justify its regulatory pathway, often referencing both biologics and device criteria. Ultimately, the successful development of MSC-EVs therapeutics will depend on interdisciplinary regulatory guidance and advanced analytical techniques, including nanoparticle tracking and microfluidics-based QC tools, to meet both safety and efficacy expectations[11,51].

Figure 7 illustrates the process of generating therapeutic EVs from MSCs, highlighting their immunomodulatory and reparative roles in ischemic and inflammatory conditions. The workflow encompasses MSC expansion, controlled bioreactor culture, and downstream concentration via TFF. Remaining challenges include developing reproducible manufacturing processes, meeting evolving regulatory standards, establishing robust potency assays, and implementing consistent criteria to ensure product quality and clinical efficacy.

Figure 7 Overview of mesenchymal stem cell-derived extracellular vesicle production, therapeutic applications, and key barriers to translation. This schematic summarizes the pathway from mesenchymal stem cells (MSCs) to the generation of therapeutic extracellular vesicles. MSC-EVs3*6 mediate immunomodulatory and reparative effects applicable to ischemic and inflammatory disorders. Scalable manufacturing requires upstream MSC expansion, controlled bioreactor cultivation, and downstream concentration via tangential flow filtration. Critical obstacles that remain include the establishment of reproducible manufacturing workflows, adherence to evolving regulatory expectations, development of reliable potency assays, and implementation of standardized criteria to ensure consistent product quality and clinical performance. MSC: Mesenchymal stem cell; TFF: Tangential flow filtration.

Alignment and standardization of GMP practices for ATMPs

Global efforts are progressing to harmonize GMP standards for ATMPs, yet notable differences remain between jurisdictions. For instance, PIC/S released Annex 2A in 2021 to align ATMP requirements with EU regulations, offering comprehensive guidance on production facilities, process controls, and operational practices[12]. This aligns with EMA’s 2017 GMP addendum and aims to foster greater international consistency. Concurrently, ICH continues to refine quality guidelines, such as the ICH Q5A(R2) update on viral safety (effective June 2024), which establishes more rigorous criteria for biotechnology-derived products[27]. EMA and PIC/S are also updating joint chapters, including chapter 1 on pharmaceutical quality, to ensure regulatory coherence[12]. Despite these harmonization efforts, variations persist: Some authorities treat cell therapies as full “biological drugs” requiring GMP compliance, while others allow certain minimally manipulated tissues to follow less stringent rules. WHO TRS 1048 Annex 3 encourages reliance on established frameworks to minimize regulatory divergence, though its guidance is not legally enforceable[15].

Overall, these developments suggest that while significant strides have been made toward harmonization, full global alignment of ATMP standards is not yet achieved. The creation of harmonized annexes and updated ICH guidance indicates progress, but sponsors must still navigate region-specific requirements, especially in countries outside PIC/S or ICH membership. Practically, many manufacturers adopt the strictest applicable standard - typically the EU ATMP GMP - for process design and subsequently adapt for other regulatory contexts. Over time, coordinated initiatives from PIC/S and WHO point toward an increasingly unified regulatory environment[12,15]. In the meantime, developers are encouraged to proactively reference international frameworks, including ICH Q9 on risk management, PIC/S guidance, and International Organization for Standardization consortium standards, to anticipate and address potential regulatory discrepancies across different regions.

Application of AI and digital platforms in the development of ATMPs

AI and ML hold significant potential to transform process monitoring, QC, and innovation in ATMP manufacturing. Although their widespread application in cell and gene therapy remains at an early stage, preliminary research indicates promising capabilities. For example, deep learning-based image analysis can assess stem cell differentiation directly from microscope images, enabling non-invasive, label-free evaluation of potency. Recent reviews highlight that structured analytical frameworks for AI-enabled quality monitoring are beginning to emerge within stem cell culture systems[1,60]. On the regulatory side, the FDA’s 2025 draft guidance encourages the adoption of advanced technologies under programs such as RMAT, signaling receptivity to data-driven approaches. Industry conferences, including ISPE Pharma 4.0 (2025), emphasize AI’s potential to digitize and enhance PAT for cell and gene therapies[77-79]. While formal guidance on AI validation in GMP environments is still forthcoming, regulatory agencies including EMA and FDA are actively assessing AI/ML lifecycle considerations.

These observations support the view that AI functions as a key enabling technology for next-generation ATMP production. Its impact is twofold: First, AI-powered PAT can accelerate batch release, detect anomalies in bioreactors, and improve process consistency, reducing both costs and failure rates; second, in silico modeling approaches, such as digital twins, enable virtual optimization of manufacturing processes before laboratory implementation. Fully realizing this potential requires extensive data collection and robust validation of AI models. At present, most implementations remain at the proof-of-concept stage. As noted in ISPE commentary, AI is poised to redefine quality management throughout the product lifecycle[60,63]. Looking ahead, regulatory frameworks are likely to incorporate AI into quality risk management strategies, for example through updates to ICH Q9. In the meantime, developers are advised to pilot AI-driven tools while maintaining traditional GMP oversight to ensure regulatory compliance and product safety.

Economic considerations represent a significant practical hurdle in ATMP manufacturing: Downstream processing and supply-chain expenses often constitute 50%-70% of total cost of goods, while the use of chemically defined, xeno-free media further elevates upstream costs[46]. Reducing these costs relies on strategies such as process intensification through high-density perfusion, implementation of closed and automated downstream workflows, and supply-chain optimizations including platform-based MCBs and scale-out production models to distribute fixed costs across multiple products[45].

TRANSLATIONAL PROGRESS, CHALLENGES, AND FUTURE DIRECTIONS

Summary of key advances

The translation of stem cell therapies and exosome-based products from laboratory research to clinical application represents an exciting scientific frontier, yet it is accompanied by significant regulatory and manufacturing challenges. Advances in scalable bioprocessing, closed automated systems, and AI-driven analytics offer tangible solutions to improve production robustness and reproducibility[3], while evolving regulatory frameworks aim to safeguard safety and efficacy, exemplified by iPSC tumorigenicity assessments[18] and standardized potency criteria[16].

Current translational limitations

Despite progress, substantial efforts remain: Harmonization of international standards (e.g., ICH, WHO, International Organization for Standardization) must keep pace with technological innovation; emerging quality attributes, such as RNA signatures in EVs, may require formal regulatory recognition; and ensuring global patient access depends on regulatory convergence across regions. Nevertheless, real-world patient access is markedly unequal, as the vast majority of approved ATMPs and ongoing clinical trials are concentrated in high-income countries. The exceptionally high cost of these therapies - frequently reaching hundreds of thousands to millions of dollars per patient - together with insufficient healthcare infrastructure, substantially limits their implementation in low- and middle-income settings[80].

Future perspectives

Success will depend on an integrated, science-driven strategy, including implementation of risk-based manufacturing per ICH Q9, utilization of closed automated platforms, and proactive alignment with evolving global regulations. Early engagement with multiple regulatory authorities (e.g., FDA, EMA, PMDA) through mechanisms such as scientific advice is essential, and investment in platform technologies - such as well-characterized MCBs and advanced analytical tools - can optimize R&D efficiency across multiple products. Continued exploration of emerging approaches, including synthetic biology for safe cell reprogramming, high-fidelity genome editing designed to reduce off-target effects associated with CRISPR technologies[81], ML for process monitoring and control, and innovative clinical trial designs, will further accelerate the field.

CONCLUSION

The widespread clinical adoption of stem cell-derived therapies and exosome products will depend on coordinated advances in manufacturing, analytical technologies, and regulatory alignment, alongside strategic efforts to reduce costs and improve global accessibility. By integrating risk-based process control with technological innovation and proactive regulatory engagement, the field can advance toward safe, reproducible, and more equitably available regenerative medicine solutions.