Challenges in the processing and preservation of adipose-derived stem cell subpopulations for clinical use

1. Introduction

The beneficial roles of distinct adipose-derived stem cell (ASC) immunophenotypical subpopulations, defined by the unique combination of cell surface markers, have been investigated in various therapeutic contexts [1]. For example, the varied differentiation potentials of ASC subpopulations, such as CD29^pos, CD34^pos, CD90^pos, CD34^posCD90^pos, can be selected for targeted treatments – bone, cartilage, or vascular tissue regeneration [2–8]. Subpopulations with potent immunomodulatory or angiogenic properties, such as CD248^pos, CD106^pos enhance the overall regenerative capacity of ASCs, making them more effective in treating complex wounds and inflammatory conditions [9,10]. Based on these promising outcomes, ASC subpopulations will be at the forefront of precise treatment in personalized interventions.

Extensive studies have investigated the preparations of optimal ASC products for clinical use, which focus on key processes such as harvest, expansion, and cryopreservation. In contrast, preparing ASC subpopulations involves different steps, as it requires identifying and sorting the targeted populations together with the subsequent expansion and cryopreservation. However, the effects of these procedures on ASC subpopulations remain poorly understood. Several crucial research questions remain: Are the sorted populations homogeneous enough to achieve the specific or expected clinical outcomes? Are there simpler or more efficient ways to identify or sort these populations? In clinical applications, additional expansion and cryopreservation are necessary – how do these procedures impact the immunophenotypes and functions of sorted ASC subpopulations? Moreover, do the changes in immunophenotype, probably caused by the additional steps, inevitably lead to predetermined functional changes in sorted ASC subpopulations?

The success of these subpopulation-based treatments depends highly on a thorough understanding of the cellular composition within the ASC subpopulations, as well as the establishment of reliable methodologies for accurately identifying and targeting desired subpopulations. Additionally, the development of scalable manufacturing and cryopreservation techniques that preserve the functions of the ASC subpopulation is essential. It is also imperative to understand how closely the functions are associated with specific immunophenotypes.

Therefore, I will analyze the aforementioned research questions. Of note, due to the limited number of studies available, my discussion will focus on presenting ideas and potential solutions based on currently available evidence. The primary goal here is to encourage further reflection, dialogue, or research on these topics.

1.1. Heterogeneity of supposedly “homogenous” ASC subpopulations

Currently, flow cytometry is widely used to analyze ASC immunophenotypes and sort specific subpopulations. The unique combination of surface markers provides valuable insights into cell properties. Therefore, how ASC subpopulations behave by analyzing the presence or absence of particular epitopes has been examined, aiming to clarify the relationship between immunophenotypic composition and cellular functionality. Of note, ASC immunophenotypic subpopulations selected by the most of current studies were defined by single epitopes or rather limited CD marker combinations. Although these approaches result in more homogeneous cells than unsorted crude ASC cultures, they still contain various subpopulations. In the previous study [11], the composition of the ASC immnunophenotypical subpopulations defined by the combination of CD73, CD90, CD105 on the primary culture was analyzed prior to sorting. The study demonstrated that when sorting was performed based solely on CD105^pos (87.74% of cells), the resulting subpopulation still comprised four distinct sub-subpopulations, CD105^posCD73^negCD90^neg (4.56%), CD105^posCD73^negCD90^pos (43.56%), CD105^posCD73^posCD90^neg (1.03%), and CD105^posCD73^posCD90^pos (38.63%). In contrast, sorting based on CD90^pos and CD105^pos together (39.66%), resulted in two subpopulations, CD105^posCD73^posCD90^neg (1.03%), and CD105^posCD73^posCD90^pos (38.63%). When all three markers were used, only a single subpopulation, CD105^posCD73^posCD90^pos (38.63%), was identified, with no further subpopulations. These observations suggest that utilizing a greater number of markers can effectively reduce heterogeneity within ASC populations. This was also validated by another study [12] which demonstrated that employing a panel of up to eight markers achieved more homogeneous cell populations by defining a more comprehensive co-expression profile.

Here, single-cell RNA sequencing or mass spectrometry are proposed to visualize the population composition of sorted ASC subpopulations and provide a comprehensive understanding of their heterogeneity and functional characteristics. Together with an array of functional assays, these methods will validate the extent to which the heterogeneity of the ASC subpopulation affects the final biological outcomes.

1.2. Technical limitations brought by fluorescence-activated cell sorting

To enhance the homogeneity, a higher number of surface markers will be selected, which will undoubtedly introduce considerable technical challenges. The need for advanced instrumentation and sophisticated analytical methods would be time-consuming and costly. Additionally, more surface markers introduced in a single flow panel can greatly compromise the detection resolution. This can complicate the differentiation and identification of low-abundance cell populations. To adequately acquire these rare subpopulations, it may be necessary to prepare over 10 million ASCs before sorting, which introduces additional practical difficulties. Fluorescence-activated cell sorting (FACS), as the widely utilized method to purify populations, also faces inherent challenges. Despite a recovery rate of up to 90%, FACS cannot collect all cells from heterogeneous mixtures, resulting in additional cell loss.

Recently, several innovative techniques have emerged for purifying the ASC subpopulations, extending beyond traditional immunophenotype-based methods. Microfluidic sorting provides high precision by manipulating cells based on size or basic physical characteristics in small-scale devices [13]. High-throughput screening can automate the identification and sorting process [14], and nanoparticle-based sorting uses targeted nanoparticles for high specificity [15]. Together, these methods provide additional options, however, the choice of sorting ultimately depends on the individual research objectives.

1.3. Immunuphenotypical instability introduced by the additional expansion

Currently, cell sorting is based on early ASC cultures or SVF. However, the acquired cell number is far behind for clinical needs. Consequently, in vitro expansion of these ASC subpopulations in the laboratory is necessary to achieve the required dose. However, after plating to the flask for additional in vitro expansions, the sorted subpopulation can barely maintain the immunophenotypic profiles they had at the time of sorting [16]. In other words, the immunophenotypic profiles change with successive cell division, a finding that has also been confirmed by others [17]. The immunophenotypic profiles defined by surface markers (biomarkers) are expected to consistently associate with the functions or determine the biological effects of the sorted ASC subpopulations. As these unexpected changes in immunophenotypes arise, it remains uncertain whether the functional properties of these pre-validated subpopulations are compromised.

Surprisingly, in our previous study, despite the diminished distinction in immunophenotypic markers, the two sorted ASC subpopulations continued to exhibit notable differences in their adipogenic capacity and wound healing efficacy [16]. This suggests that functional disparities between the subpopulations persist, even as their surface markers become less distinguishable. We propose that during cell sorting, it is feasible to isolate genetically distinct populations of stem, progenitor, or precursor cells, whose functional properties are closely associated with their defined phenotypes. The original transcriptional profiles of these purified subpopulations are likely inherited by their progeny, preserving the functional potential established at the time of sorting. Consequently, regardless of the number of passages these cells undergo in culture, their functionality remains consistent, being passed down from the initially sorted cells to their descendants. The proposal requires a direct comparison of transcriptional profiles by high-throughput RNA sequencing, along with functional assessments of ASC subpopulations, both immediately after sorting and following propagation. If the above-mentioned proposal is confirmed, it can be thus concluded that phenotypic changes, brought by additional expansion, exert negligible effects on the intrinsic properties of the sorted cells. And the immunophenotypes during sorting matter most.

Due to the immunophenotype instability, the heterogeneity level of the sorted ASC subpopulation along with culture remains complicated. Previous studies demonstrate that cells during culturing become more and more homogeneous [11,12]. Therefore, it is plausible to infer that, after plating the sorted ASC populations into flasks, cells will also become homogenous. However, due to insufficient evidence, the precise pattern of this change remains challenging to determine. Future studies should perform a comprehensive analysis of subpopulation composition based on a relatively larger number of biomarkers to draw a confirmed conclusion about the heterogeneity level of the ASC subpopulation along with dynamic culture.

Besides, the use of animal-sourced antibodies may introduce immunogenic or contaminating substances into the final cell products for clinical intentions. However, through additional washing steps and enzymatic cleavage with trypsin during the additional cultures, the original antibody can be removed. Additionally, along with the multiple cell divisions that generate more cells, the original antibody-conjugated cells can be diluted to some extent. To ensure the removal, ELISA tests can quantify trace amounts of antibodies in the cell suspension, and preclinical studies can assess any elicited immune responses.

1.4. Uncertainty brought by cryopreservation

Cryopreservation of the purified ASC subpopulations is also critical for advancing large-scale, off-the-shelf cell therapies. While cryopreservation is known to preserve the overall stemness of ASCs and maintain acceptable viability and proliferation rates, it may also induce significant changes in the expression of some surface markers [18–20]. However, research on the effects of cryopreservation on ASC subpopulations remains rather limited. The current literature lacks a comprehensive analysis of how cryopreservation affects the distribution and functionality of distinct ASC subpopulations. Our previous research has revealed that cryopreservation can markedly deplete CD248-negative subpopulations [16]. This observation suggests that the cryopreservation and thawing processes may induce selective pressures, leading to a shift in the immunophenotypic composition of ASCs. Such alterations could potentially impact the functional potency of the ASCs, as the distribution of immunophenotypes is closely linked to their therapeutic efficacy. However, the extent to which these alterations affect ASC functionality remains uncertain.

Similar to studies on ASCs, comprehensive investigations into the immunophenotypes and functions of ASC subpopulations before and after cryopreservation should be undertaken and compared to determine cryopreservation effects. Additionally, it is essential to explore how different cryopreservation durations and storage conditions impact ASC subpopulations to assess the stability of their immunophenotype and functionality over time. If the initial findings are promising, further validation of the cryopreservation’s impact on ASC efficacy in vivo should be conducted. While ASC therapy studies and those on ASC subpopulations share similarities in design, it remains crucial to specifically investigate ASC subpopulations, as this is necessary to evaluate whether the selection of populations during the cryopreservation-thawing process leads to functional challenges.

Addressing these challenges in the processing and preservation of ASC subpopulations is crucial for optimizing their clinical application. A good understanding of immunophenotypic profiles during these processes will help comprehend the functional properties of the sorted subpopulations, which are essential for targeted therapeutic outcomes. Additionally, overcoming technical limitations in sorting and cryopreservation techniques will allow for the development of scalable manufacturing processes, making ASC-based therapies more accessible and cost-effective. Ultimately, resolving these issues will enhance the efficacy and personalized nature of ASC subpopulation therapy, paving the way for their broader use in treating a variety of regenerative conditions.

In conclusion, while the therapeutic potentials of ASC subpopulations are well-recognized, many challenges remain regarding the preparation and preservation of these cells for clinical use. Processes such as additional in vitro expansion and cryopreservation post-sorting, though necessary for generating sufficient cell numbers, may lead to alterations in the immunophenotypic profiles and potentially affect the functional properties of these subpopulations. Additionally, the inherent heterogeneity within ASC subpopulations must be carefully considered. Future studies that address these issues should be conducted to ensure that the desired therapeutic outcomes are consistently achieved.

Funding Statement

This paper was not funded.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Author contributions

Qiuyue Peng: Conception and design, Manuscript writing, Final approval of manuscript