Cell Processing Center Research Articles

EVALUATION OF BLOOD CELL COUNT USING THE SYSMEX XN-1000 AUTOMATIC HEMATOLOGY ANALYZER TO OPTIMIZE COLLECTION OF PERIPHERAL BLOOD PROGENITOR CELLS BY LEUKAPHERESIS

Autologous stem cell transplantation (ASCT) is a treatment modality for several diseases. Prediction of successful mobilization may be useful to optimize hematopoietic stem cell (HSC) collection. The objective was to identify characteristics that can influence the effectiveness of mobilization and create a model to predict the success of mobilization in candidates for ASCT. This was a retrospective study, and data from patient candidates for ASCT between 09/2015 and 12/2021 were analyzed. Participants were assisted in five hospitals and laboratory procedures were performed at the Cell Processing Center (CPC) of Cetebio/Fundação Hemominas, Brazil. The medical records of the participants were reviewed to obtain the following information: gender, age, weight, diagnosis, mobilization, mobilization cycle, number of previous chemotherapy regimens and previous radiotherapy. The laboratory data analyzed were CD34+ cell enumeration and pre-collection peripheral blood (PB) blood count. Enumeration of CD34+ cell was performed using the ISHAGE protocol. The blood count was performed using an automated counter (SYSMEX XN-1000 AS-01). The studied outcome, good mobilization, was defined as a viable CD34+ cell count ≥ 20/uL in the PB. Model derivation was performed using logistic regression. The level of classification accuracy of model was tested in a subset of participants. The study population included 807 patients, 413 (51%) of whom were male. The median age was 54 (1-74) years. Multiple myeloma was the most common diagnosis (58,2%), followed by lymphoma. Of the 807 participants, 74 failed in previous mobilization attempts, totaling 881 cycles of mobilizations and collection attempts. Of the 623 participants, 353 were good mobilizers. Increasing patient weight, lower mean corpuscular volume (MCV), higher nucleated red blood cells (nRBC), peripheral blood mononuclear cells (PBMC), and immature granulocyte count were significantly associated with good mobilization. In addition, participants diagnosed with multiple myeloma were two times more likely to be good mobilizers than patients with lymphoma (OR=1.92; 95%CI: 1.21 – 3.05; P=0.006). The model was applied to the validation set to identify patients who underwent apheresis (CD34+ cells count ≥ 10 μL), resulting in a sensibility of 69%, a specificity of 95%, positive predictive value of 98%, a negative predictive value of 50%. The results of our study are extremely relevant to institutions that do not have a flow cytometer and rely on other institutions to define HSC collection by apheresis. Cetebio serves nine transplant centers. The centers that perform the collection of HSC are located between 40 to 568 km away from Cetebio (place of quantification of CD34+ cells). Predicting the outcome of mobilization may allow the adoption of complementary measures, such as the use of plerixafor. Furthermore, it would better optimize the collection of CPH by apheresis. Thus, it would be possible to avoid additional collection days, reducing costs and risks related to administration of additional doses of G-CSF, catheter maintenance and prolonged hospital stay. Success in mobilization was greater in participants who underwent the first mobilization cycle and who had a diagnosis of multiple myeloma. Furthermore, higher weight and levels of nRBC, immature granulocytes, and PBMC, as well as lower levels of MCV, were associated with successful mobilization.

Induced pluripotent stem cell-derived rejuvenated cytotoxic T lymphocyte therapy for Epstein-Barr virus-associated lymphomas: application to clinical practice

Epstein-Barr virus (EBV)-associated lymphomas are common in Asia and exhibit a poor prognosis. As EBV antigens LMP1 and LMP2 are often expressed in EBV-associated lymphomas, these lymphomas should be a good target for antigen-specific cytotoxic T lymphocyte (CTL) therapy. However, CTLs continuously exposed to viral or tumor antigens often become exhausted. Antigen-specific CTLs generated from induced pluripotent stem cells are functionally rejuvenated, showing a strong antitumor effect on EBV-associated lymphomas and persistence in vivo. For feasible "off-the-shelf" therapy, we generated allogeneic EBV-specific CTLs in the cell processing center and prepared them for actual use in clinical settings.

A Phase I/II Clinical Trial of Piggybac-Modified GMR CAR-T Cell Therapy for CD116 Positive Relapsed/Refractory Myeloid Malignancies

Background: The prognosis of relapsed/refractory (R/R) myeloid malignancies remains poor, and the development of novel treatment strategies is crucial. Although chimeric antigen receptor (CAR)-modified T cell therapy for B cell malignancies has shown excellent clinical efficacy, the use of CAR-T cells for myeloid malignancies has been more challenging, partly due to the heterogeneous expression of candidate target antigens in leukemia cells and shared expression of those antigens in normal myeloid cells or progenitor cells. We previously developed the piggyBac-modified chimeric antigen receptor (CAR)-T cells targeting CD116, also known as GM-CSF receptor alpha chain (GMR) (Nakazawa Y, et al. J Hematol Oncol. 2016 and Hasegawa A, et al. Clin Transl Immunology. 2021). GMR CAR-T cells showed substantial antitumor effects against both acute myeloid leukemia and juvenile myelomonocytic leukemia. Moreover, modulation of the spacer and antigen recognition site of the CAR vector further enhanced the anti-tumor effects of GMR CAR-T cells. GMR CAR-T cells showed an acceptable safety profile with limited cytotoxicity on normal hematopoietic cells except monocytes. Based on these results, we have initiated a first-in-human clinical trial of GMR CAR-T cell therapy in March 2021 in Japan.Study Design and Methods: The study is a phase I/II, single-center, dose-escalation study with a traditional 3+3 dose-escalation design (Table 1). Maximum of 18 patients will be recruited. Primary objectives of this study are to determine the safety and severe adverse events of piggyBac-modified GMR CAR-T cells for CD116 + relapsed/refractory myeloid malignancies by assessing the dose-limiting toxicity within 28 days from the single infusion of GMR CAR-T cells.The patients with CD116 + myeloid malignancies aged more than 1 year with myeloid malignancies who experienced an induction failure or a relapse after hematopoietic stem cell transplantation (HSCT) will be recruited. CD116 is defined as positive when a CD116 relative mean fluorescence (RFI) in leukemia cells ≥ 2. RFI was calculated by dividing the MFI of samples with that of the isotype control. Major exclusion criteria are as follows, acute promyelocytic leukemia, acute graft-versus-host disease (GVHD) (Grade ≥ 2), extensive chronic GVHD, and concurrent treatment with corticosteroid (≥ 6 mg/m2). Statistical analysis will be performed when the data will be fixed.Peripheral blood mononuclear cells will be harvested from the patient by leukapheresis and then will be transduced with GMR CAR vector by piggyBac transposon system. All manufacturing process of GMR CAR-T cells is performed in Cell Processing Center in Shinshu University Hospital under good manufacturing practice conditions. The patient will be treated with lymphodepleting chemotherapy consisting of fludarabine and cyclophosphamide, followed by CAR-T cell infusion. The dose of CAR-T cells will be 3 x 10 5 and 1 x 10 6 in cohorts 1 & 2 and cohort 3, respectively (Table 1). Kinetics of GMR CAR-T cells will be determined by quantifying the GMR CAR gene in the peripheral blood using real-time PCR after the CAR-T cell infusion. All the patients will be required to receive HSCT by day 56 following CAR-T cell infusion. The primary endpoint of the study is the safety, pharmacokinetics, and efficacy of GMR CAR-T therapy (Trial registration: jRCT2033210029).Conclusion: We herein described the protocol of first-in-human GMR CAR-T cells for relapsed/refractory myeloid malignancies. By employing the optimized CAR vector and production protocol, the safety and efficacy of GMR CAR-T cells will be evaluated in this study. [Display omitted] DisclosuresSaito: Toshiba corporation: Research Funding. Yagyu: AGC Inc.: Research Funding. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

Regenerative medicine for radiation emergencies.

ABSTRACTHiroshima University is a ‘medical institution for tertiary radiation emergencies’ and a ‘medical support organization as a part of the International Atomic Emergency Agency Emergency Preparedness Response–Response and Assistance Network (IAEA EPR-RANET)’. To establish a system of regenerative medicine for radiation emergencies with treatment by implantation of various types of cells derived from induced pluripotent stem (iPS) cells, it is necessary to establish methods of defense against and treatment for radiation-induced damage from nuclear power plant accidents and nuclear terrorism. It is also necessary to develop cell therapy, cellular repair technology and regenerative biotechnology as regenerative medicine for radiation emergencies. Such applications have not been established yet. To develop a regenerative medical system, by using the existing one, for radiation emergencies, we will attempt to manage the cell-processing center to establish a safe and secured iPS cell bank for radiation medicine. By using this iPS cell bank as the central leverage, we will develop an education program for radiation emergency medicine and construct a network of regenerative medicine for radiation emergency medicine.

A novel method for processing adipose-derived stromal stem cells using a closed cell washing concentration device with a hollow fiber membrane module

Cell-assisted lipotransfer (CAL) is an advanced lipoinjection method that uses autologous lipotransfer with addition of a stromal vascular fraction (SVF) containing adipose-derived stromal stem cells (ASCs). The CAL procedure of manual isolation of cells from fat requires cell processing to be performed in clean environment. To isolate cells from fat without the need for a cell processing center, such as in a procedure in an operation theater, we developed a novel method for processing SVF using a closed cell washing concentration device (CCD) with a hollow fiber membrane module. The CCD consists of a sterilized closed circuit, bags and hollow fiber, semi-automatic device and the device allows removal of >99.97% of collagenase from SVF while maintaining sterility. The number of nucleated cells, ASCs and viability in SVF processed by this method were equivalent to those in SVF processed using conventional manual isolation. Our results suggest that the CCD system is as reliable as manual isolation and may also be useful for CAL. This approach will help in the development of regenerative medicine at clinics without a cell processing center.

Variations in novel cellular therapy products manufacturing

At the frontier of transfusion medicine and transplantation, the field of cellular therapy is emerging. Most novel cellular therapy products are produced under investigational protocols with no clear standardization across cell processing centers. Thus, the purpose of this study was to uncover any variations in manufacturing practices for similar cellular therapy products across different cell processing laboratories worldwide. An exploratory survey that was designed to identify variations in manufacturing practices in novel cellular therapy products was sent to cell processing laboratory directors worldwide. The questionnaire focused on the manufacturing life cycle of different cell therapies (i.e., collection, purification, in vitro expansion, freezing and storage, and thawing and washing), as well as the level of regulations followed to process each product type. The majority of the centers processed hematopoietic progenitor cells (HPCs) from peripheral blood (n=18), bone marrow (n=16) or cord blood (n=19), making HPCs the most commonly processed cells. The next most commonly produced cellular therapies were lymphocytes (n=19) followed by mesenchymal stromal cells (n=14), dendritic cells (n=9) and natural killer (NK) cells (n=9). A minority of centers (<5) processed pancreatic islet cells (n=4), neural cells (n=3) and induced-pluripotent stem cells (n=3). Thirty-two laboratories processed products under an investigational status, for either phase I/II (n=27) or phase III (n=17) clinical trials. If purification methods were used, these varied for the type of product processed and by institution. Environmental monitoring methods also varied by product type and institution. This exploratory survey shows a wide variation in cellular therapy manufacturing practices across different cell processing laboratories. A better understanding of the effect of these variations on the quality of these cell-based therapies will be important to assess for further process evaluation and development.

Analysis of human nasal mucosal cell sheets fabricated using transported tissue and blood specimens.

Previously, we succeeded in transplanting autologous nasal mucosal cell sheets in the middle ears of 5 patients, who underwent cholesteatoma resection, which prevents recurrence of cholesteatoma in clinical settings. Current good manufacturing practice (GMP) standards for human cell cultivation requires the establishment of cell processing centers (CPC) which act as germ-free facilities. However, due to practical difficulties involved in establishing and maintaining such facilities at each individual hospital, a functional transport system is felt to be needed for the continuation of effective regenerative therapy. In the current study, nasal mucosal tissue and autologous blood obtained from 3 human volunteers were transported for over 3 h. Disinfected nasal tissues were cultured using keratinocyte culture medium, which included autologous serum prepared from blood. After 24 d, cultured nasal mucosal cells were transported for over 3 h and subsequently assessed for cell number, viability and purity. Moreover, CK4, CK8, and CK18 were analyzed the suitability of these nasal mucosal cell sheets for middle ear regenerative therapy.Overall, we confirmed that nasal mucosal cell sheets can be fabricated using transported nasal mucosal tissue and blood. This study would be contribute to establish a new regenerative therapy for clinical application, accompanied with transportation between companies and hospitals.

Qualidade dos produtos de terapias avançadas: requisitos de células extensamente manipuladas usadas em terapias celulares e em bioengenharia

Introduction: Preparations of products for advanced therapies include an extensive manipulation stage of the cell primary culture, which can lead to changes in the final cellular population. Standardization of the technique for obtaining the cellular cultures, in complying with Good Manufacturing Practices (GMP), and carrying out quality controls of process and final product, are essential to ensure patient safety and comparability of clinical outcomes. Objective: The purpose of this study was to identify the most prevalent trials cited in the scientific literature or in health standards applied to the evaluation of the quality of human primary cells, which could be inserted in the routine of the Cell Processing Centers. Method: A survey was carried out of scientific articles and health standards related to cell therapy and associated quality assays. Results: This study showed that the advanced cellular therapy products regulation do not specify the tests that should be used for quality control or, when specified, do not define acceptance or rejection ranges for the products, which makes urgent the discussion of such matter. Conclusions: In this article, we highlight the need to prepare the product in the form of a single batch that should be quality-controlled from samples representative of the whole. In addition, we mention some of the trials that define the quality of the advanced cellular therapy product, detailing the points at which they are to be performed in the flowchart.

再生医療の細胞培養施設の気流改善に関する研究 第３報 気流改善手法による局所清浄化能力

再生医療の細胞培養施設の気流改善に関する研究 第３報 気流改善手法による局所清浄化能力

再生医療の細胞培養施設の気流改善に関する研究 （第4報）リアルタイム浮遊微生物粒子測定装置を利用した環境モニタリング

再生医療の細胞培養施設の気流改善に関する研究 （第4報）リアルタイム浮遊微生物粒子測定装置を利用した環境モニタリング

Regulatory Landscape of Regenerative Medicine in Japan

Last year, Japan exercised two new acts, the Act on the Safety of Regenerative Medicine (RM Act) and Pharmaceuticals and Medical Devices Act (PMD Act). These acts have significantly changed the conditions for clinical application of regenerative medicine. The RM Act covers clinical research and medical practice using processed cells. It specifies the procedures required for clearance to administer said processed cells to humans. The RM Act also introduces a licensing scheme for cell processing centers outside medical institutions. The PMD Act, on the other hand, introduces a specific regulatory framework for regenerative medicine products. Under the PMD Act, conditional and time-limited marketing approval will be given to a regenerative medicine product after exploratory clinical trials have demonstrated probable benefit and proven safety. Combined with other measures to facilitate R&D, it is expected that these changes will accelerate the clinical application and commercialization of innovative regenerative medicine therapy.

再生医療の細胞培養施設の気流改善に関する研究 第2報 細胞加工作業周辺の局所清浄化の検討

再生医療の細胞培養施設の気流改善に関する研究 第2報 細胞加工作業周辺の局所清浄化の検討

The New Technology: Canal Wall Up Tympanoplasty with Transplantation of Tissue‐Engineered Cell Sheets

Objectives: The likelihood of recurrent retraction and adhesion of newly formed tympanic membrane is high when normal middle ear mucosa is extensively lost during intractable middle surgery. If rapid postoperative regeneration of the mucosa on the exposed bone surface can be achieved, prevention of recurrent tympanic membrane adhesion and cholesteatoma formation can be expected. The aim of this study was to develop a new method to transplant autologous cell-sheets to promote postoperative regeneration of the middle ear mucosa. Methods: We harvested 10-by-10-mm specimens of inferior turbinate mucosal tissue from the patient with acquired middle ear cholesteaoma. Tissue-engineered epithelial-cell sheets were fabricated ex vivo by culturing harvested cells for 3 weeks on temperature-responsive culture dishes in a cell-processing center (CPC) according to good manufacturing practice guidelines. After canal wall up tympanoplasty with mastoidectomy had been performed, sheets of cultured autologous cells that had been harvested with a simple reduced-temperature treatment were transplanted directly into the exposed bone surface of middle ear cavity from which normal mucosa had been absent. Results: During the cultivation, the sterile environment in the CPC was confirmed. Autologous cell sheets were successfully transplanted to human middle ear. Conclusions: This is the first clinical study approved from the Ministry of Health, Labour and Welfare in Japan. Furthermore this is a first-in-man study in the world in which cultured cells were transplanted to the human ear. This novel technology of transplantation might be an effective alternative to the surgical operation on intractable otitis media in the near future.

Bridging the gap: providing a web-based forum for education and communication in a challenging workplace environment

The Dana Farber Cancer Institute’s Cell Manipulation Core Facility (CMCF) is approved to provide clinical stem cell transplant services to all Harvard affiliated hospitals. As the second largest stem cell processing center in the world, the CMCF processes well over 900 products per year. Providing in-service education to clinical team members is particularly complex given the everyday challenges of communicating technical details, allocating time for busy staff, and providing access to restricted GMP lab space.

Regenerative Medicine for the Cornea

Regenerative medicine for the cornea provides a novel treatment strategy for patients with corneal diseases instead of conventional keratoplasty. Limbal transplantation has been performed in patients with a limbal stem cell deficiency. This procedure requires long-term immunosuppression that involves high risks of serious eye and systemic complications, including infection, glaucoma, and liver dysfunction. To solve these problems, ocular surface reconstruction using cultured limbal or oral mucosal epithelial stem cells has been successfully applied to patients. However, cell sheets must be fabricated in a cell processing center (CPC) under good manufacturing practice conditions for clinical use, and the expenses of maintaining a CPC are too high for all hospitals to cover. Therefore, several hospitals should share one CPC to standardize and spread the application of regenerative therapy using tissue-engineered oral mucosal epithelial cell sheets. Consequently, we developed a cell transportation technique for clinical trial to bridge hospitals. This paper reviews the current status of regenerative medicine for the cornea.

The Latest Advance in Hair Regeneration Therapy Using Proteins Secreted by Adipose-Derived Stem Cells

Introduction: Adipose-derived stem cells (ADSCs) that can be harvested from fat cells are one of the latest breakthroughs in the aesthetic field. In addition, basic studies have reported that ADSC conditioned medium (ADSC-CM) promotes skin and hair regeneration. We validate our novel approach, known as hair regenerative therapy, for hair growth treatment using ADSC-CM. Materials and Methods: ADSCs were cultured and expanded in hypoxic culture conditions, and ADSC-CM was collected. ADSC-CM includes various cytokines and growth factors that influence hair regrowth, to which we added buflomedyl, cysteine, coenzyme Q10, and vitamins. Protein solution from ADSC-CM was applied 4 to 6 times every 3 to 5 weeks by mesotherapy techniques such as nappage and papule injections. Satisfactory results of hair regenerative therapy in 12 women and 13 men were determined with a visual analog scale. Results: All patients experienced increased hair growth from the treatments with ADSC-CM. Four treatment sessions performed within 3 to 4 months provided especially good results. Scores on the visual analog scale increased with treatment frequency. Statistical significance was determined by Friedman's 2-way analysis of variance (P &lt; .01) and Wilcoxon's signed rank test (P &lt; .01). Discussion: ADSCs secrete cytokines, such as keratinocyte growth factor, vascular endotheliatl growth factor, platelet-derived growth factor, hepatocyte growth factor. Those cytokines and growth factor are very important for hair growth. Our new therapy with ADSC-CM does not require specialized facilities, such as a cell-processing center, and can be a valuable treatment.

Effects of long-term cryopreservation on peripheral blood progenitor cells

The long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA). We randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture. An age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity. Cryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34(+) cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.

A Safe and Efficacious Method for Transportation of Teeth to a Cell Processing Center for Clinical Trials with Pulp Stem Cells

A Safe and Efficacious Method for Transportation of Teeth to a Cell Processing Center for Clinical Trials with Pulp Stem Cells

Successful Post-Remission Therapy Using LMP1/LMP2a-Specific Cytotoxic T Lymphocytes (CTLs) in Extranodal NK/T Cell Lymphoma

Abstract 2709 Background:Extranodal NK/T-cell lymphoma (ENKTCL) is highly associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The role of the EBV in pathogenesis of this disease and EBV proteins expressed in this lymphoma provide targets for the adoptive immunotherapy with antigen-specific cytotoxic T lymphocyte (CTL) and raise the possibility of EBV-specific CTL as therapeutic strategies. This prospective pilot study is aimed to evaluate the effectiveness and safety of EBV-specific CTL as a maintenance therapy in patients with chemo-sensitive EBV positive ENKTCL. Methods:A total of thirteen adult patients with EBV positive ENKTCL were enrolled but two patients died due to disease progression during the generation of EBV-CTLs. Ultimately, eleven patients who were responded to chemotherapy were received EBV-CTLs. For generation of EBV-CTLs in vitro, mature dendritic cells(DC) derived from monocytes were pulsed with RNAs of EBV LMP1 and LMP2a antigens, and then T cells were stimulated with DCs three times for 3 weeks in Catholic GMP cell processing center. EBV-CTLs were cryopreserved for later usage and some remaining cells were analyzed. Patients completed the induction treatments including chemotherapy, radiotherapy, and/or high-dose therapy followed by autologous peripheral blood stem cell transplantation (HDT/SCT) before the infusion of EBV-CTLs and received 8 doses of 2 ×10E7 CTLs/m2. Results:Nine newly diagnosed and two chemo-sensitive relapsed patients (six male and five female) received maintenance therapy with EBV-CTLs. Median age was 47 years (range, 20–71 years). Ten patients achieved CR and one patient achieved partial response (PR) after induction therapy, and five patients including one patient in PR underwent HDT/SCT. During the maintenance therapy with EBV-CTLs, one patient dropped out of infection after 5 doses of EBV-CTLs therapy and the others completed 8 doses of EBV-CTLs. Among eleven patients, one patient relapsed 17.3 months since induction therapy. In overall, the 3-year overall survival (OS), progression-free survival (PFS) since induction therapy were 85.7±13.2%, 88.9±10.5%, respectively with a median follow-up of 25.2 months. For five patients who had HDT/SCT, DFS from SCT was 80.0±17.9% with a median follow-up of 21.0 months. Conclusion:This pilot study indicated that both LMP1 and LMP2a-specific CTLs can be effectively manufactured by stimulation with DCs in vitro from blood of patients with ENKTCL in current trial. This approach could be applied to patients with ENKTCL with safety and effectiveness. The larger prospective randomized study is needed to define the role of EBV-CTLs therapy to prevent unpredictable relapse in EBV-positive ENKTCL. Disclosures:No relevant conflicts of interest to declare.

Fabrication and Validation of Autologous Human Oral Mucosal Epithelial Cell Sheets to Prevent Stenosis after Esophageal Endoscopic Submucosal Dissection

Objectives: Human oral mucosal epithelial cells derived from 7 healthy volunteer donors were cultured in a clean room in a cell-processing center (CPC) according to good manufacturing practice guidelines. Cell culture and fabricated transplantable epithelial cell sheets were validated for treating ulcers after endoscopic mucosal dissection. Methods: The clonal growth and morphology of the human oral mucosal epithelial cells seeded on temperature-responsive surfaces were observed. During the cultivation, sterilization tests were performed to validate the environment in the CPC. To validate the purity and morphology of fabricated epithelial cell sheets, cell sheets harvested from temperature-responsive surfaces by temperature reduction were examined by histology and flow cytometry. Results: Human oral mucosal epithelial cells were successfully cultured and harvested as continuous cell sheets from temperature-responsive culture inserts without any animal-derived materials. During the cultivations, the sterile environment in the CPC was confirmed. The results of histological and flow cytometry analysis showed the high reproducibility of stratification and the purity of the fabricated human oral mucosal epithelial cell sheets. Conclusions: The method for fabricating epithelial cell sheets shown in this study was suitable for the validation for clinical trials and suggested usability of the fabricated cell sheets.