Culture Process Research Articles

Neonatal septicemia: Diagnostic challenges and the role of CRP and blood culture

Neonatal septicemia, a serious blood infection occurring within the first four weeks of life, is a significant cause of neonatal deaths. Its diagnosis is challenging due to the non-specific nature of its signs and symptoms. C-reactive protein (CRP) serves as a valuable biomarker for early detection, facilitating timely treatment and improving survival rates as a rapid adjunct to the standard blood culture process. This study explores the relationship between CRP levels and blood culture findings in diagnosing Neonatal septicemia. The objectives were to determine common organisms causing neonatal septicemia, to evaluate CRP as a diagnostic tool for Neonatal septicemia compared to blood culture results. The study examined the correlation between CRP levels and blood culture outcomes in neonates suspected of having sepsis. Blood samples were aseptically collected and inoculated in 1 set of BD BACTECTM Peds Plus/F blood culture bottles, incubated for five days using a BD BACTEC automated machine. The bacteria isolated from positive blood culture were processed using Vitek-2. CRP levels were determined using the latex agglutination card test, with positive samples further analyzed via quantitative nephelometry. Of the 321 samples, 28.97% (93) tested positive for bacterial sepsis via blood culture. Among these, 33.3% (31 cases) were , 18.3% (17 cases) were , and 11.8% (11 cases) were , with coagulase-negative staphylococci (CONS) being the most common group overall. Among the Gram-negative organisms, (8.6%) and (6.5%) were significant contributors to neonatal septicemia. CRP was positive in 82 cases with blood culture-positive samples and in 105 cases with blood culture-negative samples, underscoring its potential as a diagnostic marker for Neonatal septicemia. While CRP is a valuable biomarker for detection of Neonatal septicemia, it should not be used as a sole diagnostic tool due to its lack of specificity. CRP testing provides a presumptive diagnosis that can guide early antibiotic therapy, emphasizing its significance in neonatal care.

A deep learning approach to predict differentiation outcomes in hypothalamic-pituitary organoids

We use three-dimensional culture systems of human pluripotent stem cells for differentiation into pituitary organoids. Three-dimensional culture is inherently characterized by its ability to induce heterogeneous cell populations, making it difficult to maintain constant differentiation efficiency. That is why the culture process involves empirical aspects. In this study, we use deep-learning technology to create a model that can predict from images of organoids whether differentiation is progressing appropriately. Our models using EfficientNetV2-S or Vision Transformer, employing VENUS-coupled RAX expression, predictively class bright-field images of organoids into three categories with 70% accuracy, superior to expert-observer predictions. Furthermore, the model obtained by ensemble learning with the two algorithms can predict RAX expression in cells without RAX::VENUS, suggesting that our model can be deployed in clinical applications such as transplantation.

Early Diagnosis of Bloodstream Infections Using Serum Metabolomic Analysis

Background: Bloodstream infections (BSIs) pose a great challenge to treating patients, especially those with underlying diseases, such as immunodeficiency diseases. Early diagnosis helps to direct precise empirical antibiotic administration and proper clinical management. This study carried out a serum metabolomic analysis using blood specimens sampled from patients with a suspected infection whose routine culture results were later demonstrated to be positive. Methods: A liquid chromatograph-mass spectrometry-based metabolomic analysis was carried out to profile the BSI serum samples. The serum metabolomics data could be used to successfully differentiate BSIs from non-BSIs. Results: The major classes of the isolated pathogens (e.g., Gram-positive and Gram-negative bacteria) could be differentiated using our optimized statistical algorithms. In addition, by using different machine-learning algorithms, the isolated pathogens could also be classified at the species levels (e.g., Escherichia coli and Klebsiella pneumoniae) or according to their specific antibiotic-resistant phenotypes (e.g., extended-spectrum β-lactamase-producing and non-producing phenotypes) if needed. Conclusions: This study provides an early diagnosis method that could be an alternative to the traditional time-consuming culture process to identify BSIs. Moreover, this metabolomics strategy was less affected by several risk factors (e.g., antibiotics administration) that could produce false culture results.

Detaching cells in culture medium using forced vibration for removing a centrifugation from culture process

Detaching cells in culture medium using forced vibration for removing a centrifugation from culture process

Improving peritoneal dialysis fluid culture-positivity yield from 2022 to 2023.

Microbiological testing of peritoneal dialysis bags for peritonitis often yields culture-negative results. Culture-negative samples should not exceed > 15% according to the International Society for Peritoneal Dialysis. To reduce this issue, the addition of a blood culture bottle incubation step to the culture process was introduced at the Infection Control Services Laboratory (ICSL) of the National Health Laboratory Services (NHLS). The aim of the study was to ascertain if the change in methodology increased the culture-positivity yield and reduced the culture-negative percentage. Data from the NHLS Central Data Warehouse (CDW) were analysed to compare the culture-positive results over two periods: June-December 2022 when the non-blood culture (B/C) bottle method was used and January-July 2023 when the B/C bottle method was implemented. The non-B/C culture method yielded a 23% culture-positivity yield, whereas the B/C bottle-based method yielded a 51% culture-positivity yield. However, the culture-negative yield for the B/C bottle-based method was high at 49%. The change in dialysis bag processing in 2023 led to a more than doubling in culture-positivity yield. However, the culture-negative percentage remained high. As a result, further modifications to the methodology are needed. The study findings illustrate that the addition of the B/C bottle incubation step significantly improved peritoneal dialysis bag culture yields which directly impacts patient management.

Analysis of cell lysis for improved understanding between the shake tube and stirred tank reactor perfusion CHO cell cultures.

Shake tubes (ST) are widely employed to assist the development of the stirred tank reactor (STR) perfusion cell culture. However, cell lysis may be frequently underrestimated and lead to culture performance discrepency between these systems, rendering the ST model ineffective in designing the STR perfusion cultures. In this study, perfusion culture performance bewteen the STR and ST was investigated under various conditions with the analysis of cell lysis. Comparable performance was observed bewteen the two systems at low perfusion rates ( ≤1.0 VVD), except that the specific productivity ( ) of the STR was decreased at =0.5 VVD, which was related to product degradation by cell lysis. In contrast, significant differences in cell maintenance, metabolism, and were found at =2.0 VVD. By the analysis of the authentic cell growth and death kinetics, it was found that cell growth arrest, potentially due to the limited availability of oxygen, led to the stable cell maintenance at VCD≈90 × 106 cells/ml and altered cellular metabolism for the ST, while the continuous decline of VCD and in the STR were related to excessive cell death, subsequently ascribed to the harmful hydrodynamic stress conditions. We further demonstrated that cell lysis accounted for 57.62-76.29% of the total generated biomass in both the reactors and significantly impacted the estimation of process descriptors crucial for understanding the true cellular states. With cell lysis in sight, cell performance can therefore be accurately described and this knowledge can be further leveraged to expedite process development for the perfusion cell culture processes.

Establishment of callus induction and plantlet regeneration systems of Peucedanum Praeruptorum dunn based on the tissue culture method

BackgroundPeucedanum praeruptorum Dunn has typical stacked umbels and medicinal value; however, the lack of an effective tissue culture system for P. praeruptorum has limited the large-scale propagation of its seedlings.ResultsWe systematically established an in vitro regeneration system for P. praeruptorum using young leaves and stems as explants. Tissue culture plantlets were successfully obtained within 123 and 90 d of somatic embryogenesis and organogenesis, respectively. Combined plant growth regulators (PGRs) were optimized to promote efficient plant regeneration at each stage of the culture process. Specifically, embryogenic callus induction was superior in Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). For somatic embryonic development, the highest differentiation rates were achieved using BA, 2,4-D, and 6-furfuryl aminopurine (6-KT). Induction of organogenesis resulted in the highest differentiation rates and proliferation coefficients of buds in MS medium supplemented with BA and α-naphthaleneacetic acid (NAA). Moreover, regeneration of P. praeruptorum seedlings was achieved by adjusting the BA and indole-3-butyric acid (IBA) concentrations in 1/2 MS medium.ConclusionOur results provide a technical system for the rapid propagation of P. praeruptorum, which can facilitate germplasm improvement, resource conservation, and further genetic transformation of Peucedanum species.

Development of a protein production system using Ogataea minuta alcohol oxidase-deficient strain under reduced-methanol-consumption conditions.

Methylotrophic yeast is a useful host for producing heterologous proteins using the unique and strong alcohol oxidase 1 (AOX1) promoter, which is induced by methanol and repressed by various carbon sources. However, methanol is preferably avoided in industrial-scale fermentation given its toxicity, flammability, and explosiveness. To develop a protein production system under reduced methanol supply conditions, we attempted to characterize the AOX1 promoter induction activity by comparing between the de-repression and methanol induction conditions. This comparison is important because decreasing methanol consumption would enhance the industrial value of Ogataea minuta for heterologous protein production. For such a comparison, an alcohol oxidase-deficient (Δaox) strain was generated, with methanol only being used for AOX1 promoter induction. We also developed a culture process in a jar fermentor using the O. minuta Δaox strain under mixed feed conditions to achieve heterologous protein production comparable to that of the wild-type strain under low-methanol conditions.

Pomegranate plant tissue culture technique - a review

‌Background: The pomegranate, or Punica granatum L., is a member of the "Punicaceae" family, which also includes Punica protopunica and Punica granatum. Originating in Afghanistan and Iran, it has spread over Asia, Africa, and Europe's Mediterranean region. The traditional method of pomegranate propagation is laborious and time-consuming. It does not guarantee healthy, disease-free plants. Its shortcomings include poor success rates, extremely sluggish replication, and a one-year establishing period for new plants. Tissue culture is a technique that can be used to grow plants in all seasons without restrictions. Several pomegranate-growing nations have tried in vitro propagation to generate virus-free, similar-type plants, and it has been widely used for propagating many fruit trees. Research Purpose: The goal of this essay was to examine how to do plant tissue culture method and get familiar with the techniques. with an emphasis on the pomegranate micropropagation. The only aspect of plant tissue culture that may be able to get around these issues is in vitro technology. Therefore, there is plenty of room for employing micropropagation to multiply desired genotypes on a wide scale. Research Method: This review article was conducted based on scientific websites in Persian and English languages, including SID, Magiran, Google Scholar, Web of Science, and Library Studies, Results: Pomegranate plants multiply quickly through micropropagation, producing plants (clones) that are genetically similar. In a short amount of time, a single explant can yield thousands of plants. These plants match exactly. The study provides a standardized protocol for in vitro regeneration of nodal explants, which could be useful in large-scale production of uniform and disease-free plantlets. The process of plant tissue culture reduces the need for importing expensive plant seeds.

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation.

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Exploring the development of Chinese magic: take the Zoroastrian elements in unearthed artifacts as an example

This essay explres the developemnt of Chinese magic, specifically use the effect pf Zoroasteian element that developed in the illusions as an example. This essay highlighted the basic introduction of the chinese illusions from the Han to Tang Dynasties, and explained the reason why the self- injured performances is special from other illusions. The mian part of this essay is to challenge the common agreement in the academia about the time that Zoroastrianism was introduced to China during the Wei and Jin Dynasties, privide evidence to prove that ti is acutally came into China ealier in the Han Dynasty. The final part of this essay shows a usual process of foreign culture’s developemnt, which tansform from a formal ceremony to a entertaminet-purposed activity to make itself closer to publics.

DMS behaviors in pen culture of Sinonovacula constricta in Longhai, China

Dimethyl sulfide (DMS) released by the ocean has received widespread attention due to its role in aerosol formation and impact on global climate change. Research on DMS mainly focuses on coastal and open ocean environments, with limited studies addressing the influence of aquaculture activities on the production and release of marine DMS. Thus, we investigated whether fertilization for algae cultivation and the feeding process in pen culture of Sinonovacula constricta significantly contribute to DMS release. We found notable diurnal variations in DMS concentrations during and after the culture period in the ponds. During the post-culture period, both the concentration and flux of DMS in the ponds were higher than those during the culture period. The average flux of DMS during the culture and post-culture period were only 0.1 ± 0.1 μmol/(m2·d) and 0.4 ± 0.3 μmol/(m2·d), respectively, indicating that aquaculture activities of Sinonovacula constricta may not produce or release substantial DMS when compared with those from the coastal aeras and open oceans.

The Impact of Leadership Styles, Cultural Dimensions and Values on Academic Leaders

This study investigates the cultural dimensions, values, and leadership styles of school leaders in Indian K-12 and European schools, specifically focusing on cross-cultural differences. The objective is to explore how leadership styles and cultural orientations differ between Indian and European school leaders and to examine how these variations impact organizational culture and decision-making processes. A non-experimental quantitative research design was employed, utilizing standardized instruments such as the Multi-factor Leadership Questionnaire (MLQ-Form 6S), OCTAPACE, and the Scale of Individual Cultural Values (CV) to measure leadership styles and cultural dimensions. Data were collected from 165 Indian leaders across Punjab, Haryana, Delhi-NCR, and Uttar Pradesh, as well as 156 European leaders from Croatia, Hungary, Poland, and others, using purposive and convenience sampling methods. Independent t-tests and Discriminant Function Analysis (DFA) were used to compare the two regions' leadership styles and cultural orientations. The findings reveal that Indian school leaders predominantly demonstrate transformational and laissez-faire leadership behaviors, influenced by hierarchical structures prioritizing collective goals and authority delegation. Indian leaders scored higher on openness, trust, and collaboration dimensions, which align with collectivist and hierarchical cultural norms. In contrast, European leaders emphasized confrontation, authenticity, and individual autonomy, reflecting a preference for more egalitarian and individualistic decision-making approaches. These insights contribute to a deeper understanding of how leadership and cultural values shape educational practices in different contexts.

Beyond viability: Advancing CHO cell culture process strategies to modulate host cell protein levels

Chinese Hamster Ovary (CHO) cells are widely utilized in bioprocessing industry for monoclonal antibody (mAb) production. In many instances, challenges persist in achieving sufficient clearance of Host Cell Proteins (HCPs) in the final drug substance. While purification strategies usually offer substantial HCP clearance, certain "problematic" HCPs, particularly lipases, continue to pose significant challenges. This study investigates the accumulation of various "problematic" HCPs in CHO cell culture using transcriptomics, revealing correations between cell culture parameters and HCP level. Contrary to conventional assumptions, viability alone does not reliably predict HCP levels, with factors such as clone selection, host cell line choice, media and feed compositions significantly influencing HCP accumulation. Leveraging transcriptomics-based approaches, we demonstrate the potential of upstream process control strategies to mitigate HCP presence and improve biologic product quality. Our findings underscore the importance of considering diverse cell culture parameters in bioprocess optimization to ensure product stability and quality. While promising, further research is needed to elucidate the mechanisms underlying HCP release and propagation through downstream processing stages, emphasizing the necessity of a comprehensive integrated approach to HCP control in biologics production.

Procollagen-lysine 2-oxoglutarate 5-dioxygenases are responsible for 5R-hydroxylysine modification of therapeutic T-cell bispecific monoclonal antibodies produced by Chinese hamster ovary cells.

We present a detailed mass spectrometric analysis of three 2 + 1 T-cell bispecific monoclonal antibodies (TCB mAbs), where an unexpected +15.9950Da mass shift in tryptic peptides was observed. This modification was attributed to the occurrence of 5R-hydroxylysine (Hyl) using a hybrid LC-MS/MS molecular characterization and CRISPR/Cas9 gene deletion approach. The modification was found at various sites within TCB mAbs, with a conspicuous hot spot motif mirroring a prior observation where Hyl was mapped to the CH1-VH Fab domain interface of IgGs. In contrast to the preceding report, our structural modeling analysis on TCB mAbs unveiled substantial differences in the orientation and flexibility of motifs in immediate proximity and across the artificial CH1-VL cross Fab interface and upstream elbow segment. Utilizing a hybrid database search, RNAseq, and a CRISPR/Cas9 knockout methodology in Chinese hamster ovary (CHO) production cell lines, procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) were conclusively identified as the catalyzing enzymes accountable for the 5R-Hyl modification in TCB mAbs. To quantitatively inhibit Hyl formation in TCB mAbs, the activity of all three Chinese hamster PLOD isoenzymes needs to be depleted via CRISPR/Cas9 gene knockout. Moreover, our investigation identified cell culture iron availability, process duration, and clonal variability in CHO cells as elements influencing the levels of Hyl formation in TCB mAbs. This research offers a solution for circumventing Hyl formation in therapeutic complex mAb formats, such as TCB mAbs, produced in CHO cell culture processes, thereby addressing potential technical and biological challenges associated with unintended Hyl modification.

An Integrated Multi-Model Framework Utilizing Convolutional Neural Networks Coupled with Feature Extraction for Identification of 4mC Sites in DNA Sequences

N4-methylcytosine (4mC) is a chemical modification that occurs on one of the four nucleotide bases in DNA and plays a vital role in DNA expression, repair, and replication. It also actively participates in the regulation of cell differentiation and gene expression. Consequently, it is important to comprehend the role of 4mC in the epigenetic regulation for revealing the complications of the gene expression and their associated governing cellular operations. However, the inherent resource requirements and time constraints of the experimental procedure, present challenges to the cellular culture process. While data-driven methodologies present promising solutions to mitigate the demand for extensive experimental efforts, their performance relies on the suitability and existence of high-quality data. This study presents a multi-model framework that integrates convolutional neural network (CNN) with the distributed k-mer and embedding feature extraction techniques to enhance the identification of 4mC sites in DNA sequences. The integration of k-mers ensures the effective representation of the local sequence patterns, while the utilization of embedding enables a more holistic encoding by considering the broader context and semantics of the sequence data. Following the initial step, the obtained distributed representation of the DNA sequence seamlessly enters the CNN, triggering a crucial convolution operation wherein a set of adaptable filters systematically convolves across the sequence to detect vital local patterns. The proposed integrated multi-model framework was applied to six publicly available datasets and evaluated against the cutting-edge 4mCPred, 4mCCNN, iDNA4mC, Meta-4mCpred, DeepTorrent, 4mCPred-SVM, and DMKL-HFIS methods. The evaluation was based on accuracy, specificity, sensitivity, and Matthews Correlation Coefficient. The results demonstrated that the proposed multi-model framework outperformed the state-of-the-art methods, as well as one-hot encoding and the hybrid of one-hot & TNC features, in accurately identifying 4mC sites.

Cryopreservation of Neuroectoderm on a Pillar Plate and In Situ Differentiation into Human Brain Organoids.

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To demonstrate cryopreservation application to human brain organoids (HBOs), early stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NE) in an ultralow attachment (ULA) 384-well plate. The NE was transferred and encapsulated in Matrigel on the pillar plate. The NE on the pillar plate was exposed to four commercially available CPAs, including the PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80 °C and subsequently stored in a liquid nitrogen dewar. We examined the impact of the CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of NE into HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving the early stage HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability postcryopreservation than larger ones. An incubation period of 80 min with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400-600 μm. These cryopreserved early stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to noncryopreserved HBOs. The cryopreserved early stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

Platforms as audio discovery ecosystems: What Spotify’s podcast rankings tell us about the way platforms appropriate the format

Contemporary mediation gravitates around platforms, central structures where users and content converge. As the pivotal format for mediation through sound, intersecting culture, economy, technology and social processes, podcasting has drawn the attention of platforms which have been investing heavily on attracting/retaining producers and listeners alike, by facilitating production, securing the rights to exclusive content, and providing unique ways to interact with content. In this article we analyze Spotify’s Top Podcast rankings in Portugal, using descriptive analysis to identify and characterize content, to determine how discovery is promoted and how diverse is the content being presented to users, determining their perception of the format. We argue that content diversity is central to a format that has become mainstream within contemporary digital and audio culture, through the convergence of independent and mainstream producers in the unique content sphere of podcasting. Platforms may be regarded as enabling agents for this process, having become both audio discovery ecosystems and gatekeepers for the format.

Bio-printing method as a novel approach to obtain a fibrin scaffold settled by limbal epithelial cells for corneal regeneration

Treatment of Limbal Stem Cell Deficiency (LSCD), based on autologous transplantation of the patient’s stem cells, is one of the few medical stem cell therapies approved by the European Medicines Agency (EMA). It relies on isolating and culturing in vivo Limbal Epithelial Stem Cells (LESC) and then populating them on the fibrin substrate, creating a scaffold for corneal epithelial regeneration. Such a solution is then implanted into the patient’s eye. The epithelial cell culture process is specific, and its results strongly depend on the initial cell seeding density. Achieving control of the density and repeatability of the process is a desirable aim and can contribute to the success of the therapy. The study aimed to test bioprinting as a potential technique to increase the control over LESCs seeding on a scaffold and improve process reproducibility. Cells were applied to 0.5 mm thick, flat, transparent fibrin substrates using extrusion bioprinting; the control was the traditional manual application of cells using a pipette. The use of 3D printer enabled uniform coverage of the scaffold surface, and LESCs density in printed lines was close to the targeted value. Moreover, printed cells had higher cell viability than those seeded traditionally (91.1 ± 8.2% vs 82.6 ± 12.8%). The growth rate of the epithelium was higher in bioprinted samples. In both methods, the epithelium had favorable phenotypic features (p63 + and CK14 +). 3D printing constitutes a promising approach in LSCD therapy. It provides favorable conditions for LESCs growth and process reproducibility. Its application may lead to reduced cell requirements, thereby to using fewer cells on lower passages, which will contribute to preserving LESCs proliferative potential.