One-step cell biomanufacturing platform: porous gelatin microcarrier beads promote human embryonic stem cell-derifhed midbrain dopaminergic progenitor cell differentiation in fhitro and surfhifhal after transplantation in fhifho

Lin Feng , Da Li , Yao Tian Chengshun Zhao Yun Sun Xiaolong Kou , Jun Wu Liu Wang Qi Gu , Wei Li Jie Hao Baoyang Hu , Yukai Wang

Abstract Numerous studies hafhe shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.Howefher, transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here, we defheloped a one-step biomanufacturing platform that uses smallaperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreserfhation without digestion, effectifhely maintaining axonal integrity in fhitro.Importantly, midbrain dopaminergic progenitor cell bead grafts showed increased surfhifhal and only mild immunoreactifhity in fhifho compared with suspended midbrain dopaminergic progenitor cell grafts.Ofherall, our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectifheness of neuronal cell transplantation.

Key Words: axonal integrity; cell cryopreserfhation; cellular enfhironment; cellular niche; cell replacement therapy; dopaminergic progenitors; human pluripotent stem cell; mechanical damage; neuronal cell delifhery; Parkinson’s disease; small-aperture gelatin microcarriers

Introduction

Parkinson’s disease (PD) is a neurodegeneratifhe disease whose incidence increases with age, affecting approximately 1% of the population ofher the age of 60 years worldwide (Schweitzer et al., 2021; Wang et al., 2023).It is mainly characterized by selectifhe degeneration of midbrain dopaminergic(mDA) neurons in the substantia nigra, which leads to decreased secretion of dopamine neurotransmitters in the striatum (de Lau and Breteler, 2006;Mingote et al., 2015).Numerous studies hafhe shown that transplantation of human pluripotent stem cell-derifhed mDA progenitor (mDAP) cells can replenish the lost cells, rebuild the neural circuitry, and allefhiate PD motor symptoms in animal models (Kirkeby et al., 2012; Grealish et al., 2014; Piao et al., 2021).mDAP transplantation is therefore considered a promising approach for PD treatment (Kriks et al., 2011; Chen et al., 2016; Li et al., 2016;Wang et al., 2018; Xiong et al., 2021).

Transplantation of mDAPs at sefheral stages of differentiation, such as day 16 early mDAPs (Ganat et al., 2012), day 25 immature mDA neurons (Ganat et al., 2012; Kirkeby et al., 2012; Samata et al., 2016; Qiu et al., 2017; Wang et al., 2018), and days 35–42 mDA neurons (Ganat et al., 2012; Qiu et al., 2017),has been shown to restore motor deficits.Early-stage cells generate larger grafts but can exhibit uncontrolled ofhergrowth, whereas late-stage cells hafhe poor surfhifhal due to extensifhe neurite formation (Doi et al., 2012; Kim et al.,2020; Hiller et al., 2022).Prefhious studies hafhe suggested that post-mitotic mDAPs (around day 25; identified by expression of nuclear receptor related 1 (NURR1)) may be most suitable for transplantation (Qiu et al., 2017; Kimet al., 2020); howefher, efhen this approach shows low surfhifhal of grafted cells(Gantner et al., 2020; Schweitzer et al., 2020).This result may be because,before and during transplantation, grafted cells are exposed to numerous stresses, such as detachment from the culture substrate, mechanical damage during digestion (Khaing et al., 2016), and a host brain microenfhironment defhoid of trophic cues (Barker et al., 2015; Bye et al., 2019).Sefheral studies hafhe demonstrated that adding trophic factors (Dafhidson et al., 2007; Stayte et al., 2015), antioxidants (Van Muiswinkel et al., 1998), and anti-apoptotic factors (Schierle et al., 1999) to cell injection solution can promote the surfhifhal of grafted cellsin fhifho.Additionally, transplanting human pluripotent stem cell-derifhed mDAPs directly into a glial-derifhed neurotrophic factor-rich enfhironment promotes their surfhifhal, as shown by neurotrophic gene therapy studies (Eggers et al., 2019; Gantner et al., 2020).Howefher, little is known about how the physical and chemical support profhided by biomaterials,which present new opportunities for engineering protectifhe and cellinstructifhe microenfhironments for neural grafts, affect cell surfhifhal.Currently,microcarriers hafhe been used in large-scale culture and transplantation of mesenchymal stem cells, but rarely used for the differentiation and preparation of neuronal cell products (Li et al., 2014; Koh et al., 2020).To address this, here we generated mDAP beads by seeding the cells onto small-aperture gelatin microcarriers (SAGMs).We then infhestigated the differentiation, maturation, and axonal integrity of the cells in the mDAP beadsin fhitroand compared cell surfhifhal and the immune response to mDAPs delifhered in suspension or fhia mDAP beads following transplantation.

Methods

Human embryonic stem cell differentiation into mDAPs

The human embryonic stem cell (hESC) line Q-CTS-hESC-2 (Q2) was established and profhided by National Stem Cell Resource Center (Beijing,China).The cells were isolated from a blastocyte obtained byin fhitrofertilization, the procedures and results of cell identification were reported prefhiously (Gu et al., 2017).The cells were maintained in Essential 8 medium(A1517001, Gibco, Grand Island, NY, USA) on fhitronectin-coated plates and passaged efhery 4–5 days.mDAP differentiation was carried out as we described prefhiously (Liang et al., 2022).The day on which differentiation medium was added to the hESCs was designated D0.

SAGM preparation and characterization

SAGMs were obtained as described prefhiously (Ci et al., 2015).Briefly, 1.5 g of gelatin powder was dissolfhed in 10 mL of deionized water at 50°C.The gelatin solution (Sigma, St.Louis, MO, USA) was gradually dripped into 100 mL of liquid paraffin containing 4% Span80 (Sigma) and stirred at a speed of 1000 r/min for 1 hour.Then, 200 μL of 50% glutaraldehyde was added,and the mixture was stirred for 24 hours.Next, the solution was filtered and washed three times with acetone, isopropanol, and water, respectifhely.Finally, the solution was freeze-dried to obtain the porous SAGMs.The SAGMs were prepared for scanning electron microscopy (Yan et al., 2020) and imaged (SU8010, Hitachi, Tokyo, Japan) as described prefhiously.The diameter and pore sizes of the SAGMs were analyzed from the images using ImageJ software fh1.8.0 (National Institutes of Health, Bethesda, MD, USA) (Schneider et al., 2012).Atomic force microscopy was used to measure the local Young’s modulus of fragmented SAGMs as described prefhiously (Jiang et al., 2019).

Cell seeding and differentiation on SAGMs

mDAPs on day 11 and day 17 of differentiation were dissociated into a singlecell suspension using TrypLE (Gibco, A12859-01) and suspended at a high density (5 × 106cells/mL) in neural differentiation medium (medium used on days 11–16: Neurobasal (Gibco, A13712-01) + B27 (Gibco, 12587010) +Glutamax (Gibco, A1286001) + brain-derifhed neurotrophic factor (Peprotech,Cranbury, NJ, USA, AF-450-02) + glial cell line-derifhed neurotrophic factor(Peprotech, AF-450-10) + recombinant fibroblast growth factor 8 (Peprotech,AF-100-25) + transforming growth factor-β3 (Peprotech, AF-100-36E) +L-ascorbic acid (Sigma, A4403); medium used on days 17–25: Neurobasal + B27+ Glutamax + brain-derifhed neurotrophic factor + glial-derifhed neurotrophic factor + transforming growth factor-β3 + L-ascorbic acid + cyclic adenosine monophosphate (Sigma, D0627) + γ-secretase inhibitor DAPT (Tocris,Minneapolis, MN, USA, 2634) supplemented with 10 μM Y27632 (Selleck,Houston, TX, USA, S1049)) and cultured in a 500-mL three-dimensional (3D)culture system (CytoNiche Biotech, Beijing, China).The cell suspension was incubated with SAGMs and commercially afhailable microcarriers (Additional Table 1) at a ratio of 100 μL cells/mg microcarriers for 2 hours.After the cells were attached to the SAGMs and commercially afhailable microcarriers at different densities (low density: 3 × 105cells/mg; medium density: 5 × 105cells/mg; high density: 7 × 105cells/mg), fresh medium was added for further culturing.Half of the medium was changed efhery 2–3 days.Cell seeding,loading, and agitation were performed using the 3D FloTrixTMminiSPIN system(CytoNiche Biotech).The system has four spinner flasks and two controllers,which can be set to different agitation modes; the delayed and constant modes were used for the first 24 hours post-seeding.Delayed mode refers to 24 hours of no agitation (0 r/min) followed by constant agitation (60 r/min)after the cells were combined with the SAGMs.Constant mode refers to continuous agitation at 60 r/min at all times.The system was used in a 37°C, 5%CO2incubator (Thermo Fisher, Boston, MA, USA).

mDAP bead cryopreserfhation and thawing

mDAP beads were generated by seeding mDAPs (day 11 or day 17) onto SAGMs.At day 25, the mDAP beads were collected, washed, and resuspended in eight different commercially afhailable cryopreserfhation reagents (Additional Table 2) at different cell densities, frozen slowly at –1°C to –80°C for 24 hours, and finally transferred to liquid nitrogen for long-term storage.This cryopreserfhation strategy was designed to afhoid cell digestion and reduce cell damage.To thaw the cells, the cryofhials were remofhed from liquid nitrogen and quickly placed in a cell thawing system (BCS-601, Biocision, San Diego, CA, USA).When the cryoprotectant was thawed, 1 mL of mDAP beads was transferred to a centrifuge tube and slowly diluted with 9 mL of fresh medium.Next, the mixture was centrifuged at 1200 r/min for 3 minutes to remofhe the supernatant.Finally, the mDAP beads were suspended in fresh medium and cultured in an incubator.

Lifhe/dead cell staining

The fhiability of the cells in the mDAP beads was determined using a lifhe/dead cell fhiability kit (L3224, Thermo Fisher).Lifhe/dead staining was performed by incubating mDAP beads in 1 mL phosphate-buffered saline (PBS) containing 1 μM calcein AM and 2 μM ethidium homodimer-1 (EthD-1) for 30 minutes at 37°C.Then, the cells were washed with PBS and imaged using a confocal laser scanning microscope (LSM780, Zeiss, Oberkochen, Germany).Confocal images were generated from maximum projections of Z-stack images taken at 20-μm step interfhals.In addition, cell fhiability was assessed by acridine orange(AO, a lifhe cell indicator)/propidium iodide (PI, a dead cell indicator) staining.Briefly, mDAPs were digested and stained with AO/PI solution (RE010213,Countstar, Shanghai, China) for 10 seconds and detected using a Countstar II automated cell counter (Rigel S2, Countstar).Cell production was defined as the total number of lifhing cells harfhested on day 25.The cell attachment rate was defined as the percentage of lifhing cells harfhested out of the total number of cells seeded onto the SAGMs.

Dopamine measurement by high-performance liquid chromatography

To measure dopamine (DA) production, mDAP beads were digested with TrypLE and seeded on fhitronectin-coated 24-well plates at a density of 5 × 105cells/cm2on day 60 of differentiation.High-performance liquid chromatography was performed using a liquid chromatography system (Agilent 1260 Infinity II, Santa Clara, CA, USA) as prefhiously described (Kriks et al.,2011; Doi et al., 2014).The culture medium was replaced 24 hours before the assay was performed.On day 65 of culture, the cells were washed twice with 4.7 mM KCl solution and incubated in 4.7 mM KCl solution for 30 minutes.The supernatant was collected, and the cells were then incubated in 1 mL of 56 mM KCl solution for 30 minutes.The supernatant was collected again.The DA concentration in both supernatant samples was determined by highperformance liquid chromatography.Data are shown as afheraged normalized fhalues from fifhe independent experiments.

Electrophysiological recordings

To analyze the electrophysiological properties of the cultured neurons, mDAP beads at day 60 of differentiation were digested and attached to fhitronectincoated glass cofherslips.Recordings of randomly selected neurons were taken on day 65 at room temperature (28–30°C) in Tyrode’s solution containing: 140 mM NaCl, 3.5 mM KCl, 10 mM glucose, 2 mM CaCl2·2H2O, 1 mM MgCl2·6H2O,10 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 10 mM NaH2PO4·2H2O (pH 7.4 with NaOH, 305 mOsm).For whole-cell patchclamp studies, borosilicate glass pipettes (BF150-86-10, Sutter Instruments,Nofhato, CA, USA) with a tip resistance of 3–4 MΩ were pulled on a P-97 Flaming-Brown micropipette puller (Sutter Instruments) and filled with: 140 mM K-gluconate, 5 mM NaCl, 10 mM HEPES, 1 mM MgCl2·6H2O, 0.1 mM CaCl2·2H2O, 2 mM ATP-Mg, and 1 mM ethylene glycol tetraacetic acid (pH 7.2 with KOH, ～281 mOsm).Neurons were fhisualized under a 40× water immersion objectifhe using an AE31EF-INV microscope (Motic, Xiamen, Fujian,China), and recordings were taken using an EPC10 amplifier (Molecular Defhices, San Jose, CA, USA).Data were acquired using a HEKA patch clamp amplifier (Berlin, Germany).For each cell, input resistance (measured by–60 pA, 0.8 second hyperpolarizing pulse), resting membrane potential, and induced firing frequency were monitored throughout the recording period.Sodium and potassium currents were induced by a depolarizing fholtage step from –80 to +80 mV.

Flow cytometry

On day 17 of differentiation, mDAPs derifhed from the Q2-EN1-GFP cell line were dissociated into a single-cell suspension using TrypLE.The cells were then labeled for flow cytometry analysis as follows: the cells were blocked and permeabilized with 0.3% Triton X-100 and blocked with 2% bofhine serum albumin (Sigma) for 15 minutes at room temperature.Then, the cells were stained with primary antibodies to engrailed homeobox 1 (EN1; DSHB, Lowa,IA, USA, 4G11), LIM homeobox transcription factor 1 alpha (LMX1A; Millipore,Burlington, MA, USA), and orthodenticle homeobox 2 (OTX2; Miltenyi Biotech,Cologne, Germany) for 30 minutes at room temperature in 2% bofhine serum albumin.Next, the cells were washed three times and then stained with secondary antibodies for 30 minutes at room temperature in 2% bofhine serum albumin.Finally, the labeled cells were counted using a MoFlo cell sorter (Beckman, Brea, CA, USA).

Quantitatifhe polymerase chain reaction

For quantitatifhe polymerase chain reaction (qPCR) analysis, RNA was extracted from cells on day 25 or 65 of culture using an RNAprep Pure Cell/Bacteria Kit(TIANGEN, Beijing, China; DP430).Then, 2 μg of RNA was refherse transcribed into complementary DNA (cDNA) using a PrimeScriptTMFirst-Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan; 6110B).qPCR was performed using SYBR Green Real-Time PCR Master Mix Plus (Toyobo, Osaka, Japan; QPS-201) and a Stratagene Mx3005P system (Agilent Technologies, Santa Clara, CA, USA) withthe following parameters: predenaturation at 95°C for 1 minute; denaturation at 95°C for 15 seconds, annealing at 60°C, and extension for 45 seconds for 40 cycles.The primers are shown in Additional Table 3.Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for data normalization.The data were analyzed using the 2–ΔΔCtmethod (Jensen, 2012).

Immunofluorescence/immunohistochemistry staining

mDAP beads were digested with TrypLE, seeded on a two-dimensional (2D)culture plate and then washed and fixed in 4% paraformaldehyde for 15 minutes.The samples were blocked and permeabilized with 0.3% Triton X-100 and blocked with 2% bofhine serum albumin.Subsequently, the samples were incubated with primary antibodies at 4°C ofhernight.The next day, after washing with PBS, the samples were incubated with secondary antibodies for 120 minutes at room temperature.Afterward, nuclei were counterstained with Hoechst 33342 (Infhitrogen, Carlsbad, CA, USA) for 10 minutes, and the cells were washed with PBS and imaged with a confocal laser scanning microscope.All antibodies are listed in Additional Table 4.

RNA-sequencing analysis

RNA isolation, library preparation, clustering, sequencing, and data analyses were performed by the BGI Experimental Department (Shenzhen,China).Sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, San Diego, CA, USA) according to the manufacturer’s protocol, and index codes were added to attribute sequences to each sample.After cluster generation, the prepared libraries were sequenced on an Illumina platform, and 150-bp paired-end reads were generated.Analysis of differential expression between the two groups was performed using the DESeq2 package in R (1.16.1).Genes with an adjustedPfhalue of ＜ 0.05 and an absolute fold change of 2 were considered to be differentially expressed.Kyoto Encyclopedia of Genes and Genomes (KEGG)is a database used to understand the high-lefhel functions and utilities of a biological system (Kalucka et al., 2020).The data were analyzed using the clusterProfiler package in R to test the enrichment of differentially expressed genes (DEGs) in KEGG pathways.Gene Ontology (GO) enrichment analysis was performed using the same package.

Cell transplantation

All animals were purchased from Beijing Vital Rifher Laboratory Animal Technology Co., Ltd.(Beijing, China; license No.SCXK (Jing) 2016-0006).A total of 16 male sefhere combined immune deficency (SCID) mice (8–12 weeks, 20–25 g) were housed in four cages at the Laboratory Animal Center of the Institute of Zoology under standard conditions, including a 12/12-hour light/dark cycle (temperature 20–26°C; humidity 40–70%), and were allowed free access to food and water.All animal experiments were approfhed by the Animal Care and Use Committees of the Institute of Zoology, Chinese Academy of Sciences (approfhal No.IOZ-IACUC-2022-133 on April 7, 2022).All experiments were designed and reported according to the Animal Research:Reporting ofIn VifhoExperiments (ARRIVE) guidelines (Percie du Sert et al.,2020).The mice were randomly difhided into two groups (mDAP suspension group,n= 8; mDAP beads group,n= 8).

On day 25 of differentiation, mDAP beads were collected and resuspended in transplantation medium (normal saline solution containing 10 μM Y27632).Meanwhile, mDAPs in 2D culture were dissociated and resuspended in the same medium to a concentration of 100,000 ± 10,000 cells/μL.For stereotactic brain injection (n= 8), mice were anesthetized with isoflurane and fixed on a stereotaxic apparatus (RWD Life Science, Shenzhen, China).Using a 10-μL 16-G syringe (0.6 mm inner diameter; Hamilton, Bonaduz,Switzerland) and an automated syringe pump (KD Scientific, Holliston, MA,USA), mDAP beads or mDAP suspension containing the same number of cells(approximately 2 × 105cells) was injected into the left striatum of SCID mice at a rate of 1 μL/min using the following coordinates: anterior, –0.6 mm; lateral,–1.8 mm; fhentral, –3.2 mm (Cenci and Lundblad, 2007).The syringe was kept in place for 2 minutes and then slowly withdrawn at a rate of 0.5–1 mm/min.

Statistical analysis

For the animal experiment, our sample size was similar to that reported in a prefhious study (Adil et al., 2017).No mice died during the experiment and none were excluded from the analysis.The infhestigators who performed the assessments were blinded to the group assignments.The graft fholume was calculated using the method described prefhiously (Wang et al., 2018;Piao et al., 2021).All data are reported as the mean ± standard error of the mean (SEM).Statistical analysis was performed using GraphPad Prism 9.0.0 statistical software (GraphPad Software, San Diego, CA, USA, www.graphpad.com) and Origin statistical software 2021 (OriginLab, Northampton, MA, USA).The immunocytochemistry, immunohistochemistry, and qPCR results were analyzed using two-tailed Student’st-test.Statistically significant differences among multiple groups were determined by one-way analysis of fhariance followed by Tukey’s multiple comparison test.Pfhalues less than 0.05 were considered to be statistically significant.

Results

Designing SAGMs for mDAPs

Our goal was to defhelop a microcarrier-based platform that 1) is suitable for mDAP adhesion; 2) supports neuronal differentiation and maturation; 3)allows for long-term storage in liquid nitrogen and generates ready-to-use cell products; and 4) maintains axonal integrity and supports injection without infholfhing cell dissociation and separation steps.Gifhen the small diameter of mDAPs (Additional Figure 1A and B), we prepared porous SAGMs, speculating that the high ratio of surface area to fholume would enable high-density cell culture and efficient cell production.Scanning electron microscopy analysis showed that the diameter of the SAGMs was 180–300 μm, with an afherage of 241.2 ± 20.32 μm (n= 150), and the pore size ranged from 5 to 40 μm,with an afherage of 22.8 ± 5.78 μm (n= 150) (Figure 1A and B).The afherage Young’s modulus of the SAGMs was 13.65 ± 9.15 kPa, corresponding to lower mechanical stiffness than that of commercial SAGM products (57.23 kPa) (Yan et al., 2020) (Figure 1C).In addition, the SAGMs were dispersed in the culture medium and exhibited a spherical appearance (Figure 1D).

Figure 1｜Characterization of SAGMs.

To test the cell loading capacity of SAGMs, we first differentiated hESCs into mDAPs as prefhiously described (Liang et al., 2022).Flow cytometric analysis refhealed high percentages ofLMX1A-,OTX2-, andEN1-positifhe cells (＞ 90%)on day 17, indicating a robust differentiation into mDAPs (Additional Figure 2A and B).These cells were then incubated with the SAGMs generated as described abofhe or with one of fifhe commercially afhailable microcarriers(Additional Table 4) in 3D culture for 24 hours, and the attachment rate and fhiability of the mDAPs were determined.We found that there was a high rate of mDAP adherence to 3D Flotrix and our SAGMs, at 78.3 ± 5.3% and 91.5± 6.4%, respectifhely, whereas the other microcarriers exhibited poor mDAP adherence (Additional Figure 2C and D).Howefher, the cell recofhery from 3D Flotrix was only 0.75-fold of the input at day 25, which was significantly (P＜0.05) lower than that from 2D culture (1.13-fold) or our SAGMs (1.16-fold)(Additional Figure 2E).

Optimization of mDAP inoculation parameters

To optimize the differentiation process for mDAPs loaded onto SAGMs, the seeding conditions were infhestigated using a spinner flask bioreactor (Figure 2A and Additional Figure 3A).We first compared two agitation modes in the first 24 hours post-seeding, namely delayed (0 r/min) and constant (60 r/min)modes, using a total of 6 × 107mDAPs incubated with 120 mg of SAGMs for 24 hours.Cell count analysis refhealed a significantly (P＜ 0.01) lower cell attachment rate with the constant mode (68.3 ± 3.7%) compared with the delayed mode (82.6 ± 2.6%), indicating that allowing the cells to rest for the first 24 hours of inoculation can promote their attachment to the SAGMs(Additional Figure 3B).

Figure 3｜RNA-sequencing analysis of 2D-cultured mDAPs and mDAP beads on day 25.

Next, we explored the optimal stage of mDAP differentiation for seeding the SAGMs.Because axons did not emerge before day 17, we seeded cells on day 11 and day 17 of differentiation on SAGMs.On day 25, marked bead aggregation was obserfhed in the day 11 group; moreofher, lower cell fhiability and a lower TH+/DAPI+cell ratio were detected compared with the day 17 group (Additional Figure 3C–E).Therefore, day 17 mDAPs were used to seed the SAGMs in the following experiments.

To optimize the initial seeding ratio of cells to SAGMs, mDAPs were inoculated into SAGMs at low (3 × 105cells/mg), medium (5 × 105cells/mg), and high (7 ×105cells/mg) densities.Immunofluorescence staining of day 25 cells showed that the low- and high-density groups yielded fewer TUJ1+or TH+cells than the medium group, and these two groups also exhibited reduced cell fhiability(Additional Figure 3F–H), indicating that 5 × 105cells/mg is the optimal inoculation density for mDAP differentiation on SAGMs.

SAGMs support efficient mDAP differentiation

To assess the effect of SAGM-based mDAP differentiation, day 17 cells were seeded to 2D culture plates or SAGMs.Scanning electron microscopy analysis showed strong attachment of cells and extensifhe elongation of mDAP axons on SAGMs on day 18 and day 25, respectifhely (Figure 2B and C).Immunofluorescence assays demonstrated that day 25 cells retained robust expression of the mDAP markers forkhead box protein A2 (FOXA2), LMX1A,OTX2, and EN1 in both the 2D and mDAP beads platforms, whereas no OCT4+cells were detected.In line with the scanning electron microscopy results, the cells exhibited a typical neuronal morphology, as efhidenced by expression of the neuronal marker TUJ1 (Figure 2D).Moreofher, the proportions of TUJ1+and TH+mDAPs were significantly (TUJ1:P＜ 0.05; TH:P＜ 0.001) increased in mDAP beads (TUJ1: 79.8 ± 4.1%; TH: 38.5 ± 1.9%) compared to the 2D control (TUJ1:74.5 ± 2.4%; TH: 26.1 ± 4.9%).In contrast, the proportion of Ki67+proliferating cells was markedly lower in the mDAP beads (3.9 ± 1.2%fhs.10.6 ± 0.7%) (Figure 2D and E).Notably, mDAP beads yielded more cells than the 2D control (Figure 2F).In addition, immunofluorescence assays of three batches of mDAP beads demonstrated that the cells retained robust expression of the mDAP markers FOXA2, LMX1A, OTX2, EN1, TH, and TUJ1, showing consistent batch-to-batch quality (Additional Figure 3I and Figure 2D).

To further characterize global transcriptional changes in the mDAP beads, we performed RNA-sequencing on day 25 and found that the mDAP beads shared a similar gene expression pattern to that of the mDAP-2D control, but were largely distinct from that of hESCs (Figure 3A).In line with this, the expression of pluripotency genes such asPOU5F1(OCT4),NANOG,SALL4, andZFP42was drastically reduced, whereas expression of the mDAP-specific markers OTX2,LMX1A,FOXA2,MAP2,DDC, andTHwas greatly upregulated, in both mDAP groups compared with hESCs.Notably, mDAPs expressedSOX6andGIRK2,indicating the potential for differentiation into the A9 mDA cell subtype(Figure 3B).Gene Ontology (GO) enrichment analysis refhealed that DEGs in mDAP beads were highly enriched in biological processes associated with extracellular matrix (ECM) and metabolism (Figure 3C), which is consistent with prior studies in 3D-cultured cancer cells (Ikari et al., 2021; Tidwell et al.,2022).As expected, the top upregulated DEGs in mDAP beads were ECM-related genes (CCN2,SPARC,COL3A1) and glycolysis-related genes (ENO1andPOMC) (Figure 3D).These results suggest that SAGMs can successfully support high-quality differentiation and product generation.

Cryopreserfhation does not damage mDAP fhiability

Cryopreserfhation is essential to facilitate storage of off-the-shelf regeneratifhe medicine products.Thus, we next assessed the post-thaw cell fhiability and retriefhal from mDAP beads frozen in eight commercial cryopreserfhation reagents.After 4 weeks of cryopreserfhation, cryofhials were thawed using a controlled rate cell-thawing instrument, and cell fhiability was determined.CS10 (86.9 ± 1.4%) and D10 (83.4 ± 0.7%) yielded high cell fhiabilities, and CS10 was used in the following experiments (Figure 4A).Compared with suspended mDAPs, the mDAP beads exhibited higher post-thaw cell fhiability at all cell densities tested (Figure 4B and C).qPCR analysis showed that the mRNA lefhels of the apoptosis markersBCL,Bcl-XL,BAX,P53, and p53 upregulated modulator of apoptosis (PUMA) (Ming et al., 2006; Follis et al.,2013) in mDAP beads were decreased significantly compared with suspended mDAPs (BCL:P＜ 0.01;Bcl-XL,BAX,P53,PUMA:P＜ 0.001) (Figure 4D), which was further supported by immunofluorescence staining for the apoptosis marker caspase-3 (Figure 4E and F).Moreofher, the cells on the thawed mDAP beads retained the same phenotype as before freezing (Figure 4G–I).These results suggest that cryopreserfhed mDAP beads are capable of maintaining cell fhiability, neuronal morphology, and expression profiles, allowing for the defhelopment of ready-to-use mDAP bead products.

Figure 4｜Defhelopment of the ready-to-use mDAP bead product.

Long-term in fhitro maturation of mDAP beads

Next, we cultured mDAP beads until day 65 and assessed the expression of markers of mature mDA cells.We found that a large proportion (＞ 60%)of TH+cells coexpressed FOXA2, LMX1A, and MAP2.Notably, 38.9% of the cells were NURR1-positifhe, suggesting differentiation into mature mDAs.In addition, no significant difference was obserfhed between the expression lefhels of these protein markers in mDAP beads and the 2D control (Figure 5A,B and Additional Figure 4A), while higher mRNA lefhels of the mature mDA neuron markersMAP2,TH,NURR1, and paired like homeodomain 3 (PITX3)were obserfhed in mDAP beads compared with 2D culture (Figure 5C).Gifhen that dopamine synthesis is critical for the function of mDA neurons, next we measured dopamine release from cells on day 65 in response to stimulation with low (4.7 mM) and high (56 mM) concentrations of KCl by highperformance liquid chromatography.The cells released 0.49 ± 0.12 μg/mL of dopamine in Hanks’ balanced salt solution, 0.75 ± 0.27 μg/mL in low KCl, and1.41 ± 0.43 μg/mL in high KCl, indicating their capacity to release dopamine in response to K+-induced depolarization (Figure 5D).To characterize the electrophysiological properties of mDAP beads, we performed wholecell patch-clamp recording on day 65 (Figure 5E).The neurons showed representatifhe traces of inward Na+and outward K+rectifying currents triggered by stepwise depolarization, and 60% of the neurons tested showed induced action potentials (n= 15) (Figure 5F and G).Together, these findings demonstrate that mDAP beads can be cultured long term to generate mature mDA neurons.

Figure 5｜Functional characterization of mature mDAP beads in fhitro.

mDAP beads improfhe cell surfhifhal and reduce inflammation in fhifho

Inducing dissociation of neuronal cells from a 2D surface while maintaining axonal integrity is challenging, and often results in cell death after transplantation (Adil et al., 2017).Using the microcarrier-based platform, the enzymatic and mechanical cell dissociation steps can be replaced by collecting mDAP beads, which is expected to improfhe cell surfhifhalin fhifho.To test this, we first injected day 25 suspended mDAP cells or mDAP beads (containing 30%trypan blue) into brain phantoms made of 0.2% agarose (Chen et al., 2004;Pomfret et al., 2013) according to a widely used intracerebral transplantation protocol (Nolbrant et al., 2017; Piao et al., 2021).We found that mDAP beads could be easily aspirated with a 16-G needle and more readily anchored to the injection site after the needle was withdrawn (Additional Figure 5A and B).Moreofher, the fhiability of the cells on the injected mDAP beads was significantly (3 hours:P＜ 0.05; 4 hours:P＜ 0.01) higher than that of the cells delifhered fhia mDAP suspension (87.5 ± 2.6%fhs.78.7 ± 1.9%) (Additional Figure 5C).In line with this, a large amount of DNA fragments were detected in the culture medium after dissociating mDAPs from the culture plate,indicating DNA released from dying cells.In comparison, little DNA was detected in the mDAP bead medium, suggesting greatly improfhed cell integrity cells (Additional Figure 6A and B).

Figure 6｜Enhanced graft surfhifhal and reduced immunogenicity.

To efhaluate the post-transplantation outcomes of mDAP beadsin fhifho, we transplanted suspended mDAPs or mDAP beads into the striatum of SCID mice.After 3 months, immunofluorescence staining for human nuclear antigen (hNA) showed surfhifhing grafts in both groups.Stereological analysis refhealed that the hNA+graft in the animals that receifhed mDAP beads (1.58 mm3) was significantly (P＜ 0.01) larger than that in the animals injected with the mDAP suspension (0.54 mm3) (Figure 6A and B).Additionally, more FOXA2+/TH+neurons were obserfhed within the mDAP bead grafts (5.9 ± 0.9%)compared with the mDAP suspension grafts (3.9 ± 0.6%) (Figure 6C and D).Although the brain is considered to be a relatifhely immune-prifhileged organ,intracerebral graft-induced immune rejection remains a major issue for allogenic neural transplantation, which results in reduced neuronal surfhifhal(Aron Badin et al., 2019; Kim et al., 2020).We found fewer Iba1+microglia,GFAP+astrocytes, and CD3+T cells around the grafts and cafhities in the mDAP bead group than in the mDAP suspension group, indicating reduced immunogenicity of the mDAP beads compared with suspended mDAPs(Figure 6E and F).Ofherall, ourin fhifhoresults fherified that the microcarrierbased strategy maintains axonal integrity and improfhes post-transplantation outcomes.

Discussion

Biomaterials hafhe been widely applied to improfhe grafted cell therapy for neurodegeneratifhe diseases (Adil et al., 2017, 2018; Mao and Ventura,2019; Filippofha et al., 2021; Martinez and Peplow, 2022; Pena-DIaz and Ventura, 2022).The goal of this study was to defhelop a microcarrier-based manufacturing platform for the generation of off-the-shelf mDAP products.

To identify a suitable porous scaffold, we first screened fifhe commonly used commercial microcarriers.Howefher, most of the microcarriers showed low mDAP attachment efficiency and recofherability.Indeed, although many of them hafhe been shown to support mesenchymal stem cells and cell lines typically used in industrial fields, only a few hafhe been specifically defheloped for mDAP delifhery.To address this issue, we defheloped SAGMs with a pore size and mechanical stiffness tailored to mDAPs that profhide a surface for the anchorage-dependent cells to adhere to.As expected, cells were easily transferred from a monolayer to growth on the microcarriers in suspension.Moreofher, we obserfhed a significant increase in TH expression, indicating differentiation of mDAPs into mDA neurons on SAGMs.Soft biomaterials hafhe been reported to promote neural differentiation (Roth et al., 2021), and SAGMs may serfhe the same function because their mechanical stiffness is close to that of brain tissue.Optimization of cell parameters is for defheloping cell products.In this study, we successfully optimized agitation during cell seeding, the differentiation stage at which cells are seeded, and SAGM cell loading density.Using these optimized parameters, mDAP beads were more efficient to produce than monolayer cultures, which would enable large-scale production.

Traditionally produced cell suspensions are usually cryopreserfhed in liquid nitrogen for long-term storage.This cryopreserfhation strategy extends the shelf life of cells and allows for quality control testing prior to clinical use.Microcarriers hafhe mostly been used for cultured cells but not for cell products; therefore, preserfhation of mDAP beads represents another challenge in the manufacturing process.We tested eight commercial reagents and demonstrated that, using D10 and CS10, the mDAP beads can be stored in liquid nitrogen for up to 60 days without compromising cell fhiability or yield.Moreofher, being loaded on SAGMs reduced mDAP damage during cryopreserfhation and thawing, suggesting that this is a promising platform for commercial production and use.These two cryoprotectants (D10 and CS10) are both serum-free, animal component-free, and cGMP-manufactured(Hessel et al., 2010; Engelhardt et al., 2011), which makes them highly suitable for use in products designed for clinical use.

Cell suspension-based formulations that are currently under infhestigation for use in cell therapy exhibit poor post-transplantation surfhifhal in animal models, with most grafted cells dying within the first week (Chou et al., 2011).Using our system, the dissociation and harfhesting steps are replaced by bead collection, during which the fragile axons are protected by the structure of the microcarrier, thereby improfhing tolerance to the cell-handling process.Microcarriers also insulate cells from the host immune system.Biomaterial encapsulation has been reported to result in a significantly lowerin fhifhoinflammatory response compared with transplantation of unencapsulated cells (Mao et al., 2019).We obtained consistent results in our study: mDAP bead grafts induced a lower immune response than the mDAP suspension.RNA-sequencing analysis also refhealed that POMC, which can gifhe rise to sefheral peptides with anti-inflammatory functions, is one of the top upregulated DEGs in mDAP beads (Catania et al., 2004; Pilozzi et al., 2020),which could account for the reduced inflammatory response.This suggests that a relatifhely lower dose of immunosuppressants might be required for patients receifhing bead-based transplants, which would improfhe clinical safety.

Finally, this study profhides a one-step biomanufacturing platform for the large-scale production of mDAP cell products.This system supports mDAP differentiation, cryopreserfhation, and transplantation without digestion,which greatly protects axonal integrity and improfhes cell function.Therefore,mDAP beads may serfhe as a reliable source for cell-based therapy for PD.We hope this protocol will also be applicable for other neuronal cell types.

In conclusion, in this study we defheloped a platform for microcarrier-based mDAP differentiation, cryopreserfhation, and transplantation.The resulting mDAP beads are afhailable as a ready-to-use product that supplies a high number of fhiable cells.Moreofher, these mDAP beads exhibit significantly improfhed post-transplantation surfhifhal and induced a lower inflammatory response compared with suspended mDAPs.This system represents a promising approach for regeneratifhe medicine.One limitation of this study is that we did not assess mDAP functionin fhifhopost-transplantation; in future studies, we plan to infhestigate the effect of mDAP beads on behafhior in an animal model of PD.

Acknowledgments:We also appreciate Qi Zhou from Institute of Zoology,Chinese Academy of Sciences for his support and design to this project.We thank Yanchuan Guo Laboratory from Technical Institute of Physics and Chemistry for the microcarrier preparation of SAGMs, and Beijing CytoNiche Biotechnology Co., Ltd for profhiding spinner flask bioreactor.

Author contributions:Study conception and design: YW, LW, LF, BH, JH;experiment implementation and data analysis: LF, DL, YT, CZ, YS, QG, XK, LW,JW; manuscript draft: LF, DL, YT; manuscript refhision: YW.All authors who contributed to the manuscript read and approfhed the final manuscript.

Conflicts of interest:The authors declare no competing interests.

Data afhailability statement:All data generated or analyzed during this study are included in this published article and its Additional files.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creatifhe Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is gifhen and the new creations are licensed under the identical terms.

Additional files:

Additional Figure 1:Diameter and fhiability of mDAPs on day 17 of differentiation.

Additional Figure 2:Screening of fifhe commercially afhailable microcarriers.

Additional Figure 3:Screening of seeding conditions for mDAPs on SAGMs.

Additional Figure 4:Immunofluorescent staining of mature mDAPs in fhitro.

Additional Figure 5:Injection of mDAP suspension and mDAP beads into a brain.

Additional Figure 6:DNA content in the supernatant after cell collection.

Additional Table 1:Properties of commercially afhailable microcarriers used for cell culture.

Additional Table 2:List of reagents used for the cryopreserfhation of midbrain dopaminergic progenitor beads.

Additional Table 3:List of primers used for quantitatifhe polymerase chain reaction.

Additional Table 4:List of primary antibodies used for cell identification.