Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations.

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system.

BACKGROUND: Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. RESULTS: Analyses of the bacterial community identified in water from BFT and "clear seawater" (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. CONCLUSION: This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Protein markers for identification of Yersinia pestis and their variation related to culture.

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C-30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

Intestinal organoid modeling: bridging the gap from experimental model to clinical translation.

The 3D culture of intestinal organoids entails embedding isolated intestinal crypts and bone marrow mesenchymal stem cells within a growth factor-enriched matrix gel. This process leads to the formation of hollow microspheres with structures resembling intestinal epithelial cells, which are referred to as intestinal organoids. These structures encompass various functional epithelial cell types found in the small intestine and closely mimic the organizational patterns of the small intestine, earning them the name "mini-intestines". Intestinal tumors are prevalent within the digestive system and represent a significant menace to human health. Through the application of 3D culture technology, miniature colorectal organs can be cultivated to retain the genetic characteristics of the primary tumor. This innovation offers novel prospects for individualized treatments among patients with intestinal tumors. Presently established libraries of patient-derived organoids serve as potent tools for conducting comprehensive investigations into tissue functionality, developmental processes, tumorigenesis, and the pathobiology of cancer. This review explores the origins of intestinal organoids, their culturing environments, and their advancements in the realm of precision medicine. It also addresses the current challenges and outlines future prospects for development.

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

STUDY QUESTION: Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? SUMMARY ANSWER: A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. WHAT IS KNOWN ALREADY: In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. STUDY DESIGN SIZE DURATION: Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. PARTICIPANTS/MATERIALS SETTING METHODS: Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 µm compared to 67.3 ± 7.2 µm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 µm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. WIDER IMPLICATIONS OF THE FINDINGS: Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

Factors affecting the quality of therapeutic proteins in recombinant Chinese hamster ovary cell culture.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the commercial production of therapeutic proteins. Fed-batch culture is widely used to produce therapeutic proteins, including monoclonal antibodies, because of its operational simplicity and high product titer. Despite technical advances in the development of culture media and cell cultures, it is still challenging to maintain high productivity in fed-batch cultures while also ensuring good product quality. In this review, factors that affect the quality attributes of therapeutic proteins in recombinant CHO (rCHO) cell culture, such as glycosylation, charge variation, aggregation, and degradation, are summarized and categorized into three groups: culture environments, chemical additives, and host cell proteins accumulated in culture supernatants. Understanding the factors that influence the therapeutic protein quality in rCHO cell culture will facilitate the development of large-scale, high-yield fed-batch culture processes for the production of high-quality therapeutic proteins.

Development of a Strategy to Improve the Stability of Culture Environment for Docosahexaenoic Acid Fermentation by Schizochytrium sp.

A stable culture environment is the key for optimal growth and metabolic activity of microorganisms, especially in marine species, and intermittent feeding during DHA production using Schizochytrium sp. generates an unstable culture environment. To investigate the effect of unstable culture environment on the cells' physiological status and DHA synthesis, fermentations with different feeding strategies were performed on the lab scale. The intermittent feeding strategy caused fluctuations of substrate concentration and osmotic pressure, which had a negative effect on cell division and product synthesis. The physiological status and metabolic level of Schizochytrium sp. were relatively stable under a continuous feeding strategy with a relatively stable substrate concentration of 20-25 g/L, which was beneficial for the efficient transformation of substrate, leading to an improvement of DHA productivity. This strategy was further applied to pilot scale, whereby the DHA content, DHA productivity, convert ratio of glucose to lipid and DHA reached 55.02%, 320.17 mg/(L·h), 24.35%, and 13.40%, respectively. This study therefore provides an efficient strategy for ensuring a stable culture environment for the production of DHA and similar metabolites. Graphical Abstract.

Design and characterization of microspheres for a 3D mesenchymal stem cell culture.

Recent studies have shown the relevance of growing mesenchymal stem cells (MSCs) in three-dimensional environments with respect to the monolayer cell culture on an adherent substrate. In this sense, macroporous scaffolds and hydrogels have been used as three-dimensional (3D) supports. In this work, we explored the culture of MSCs in a 3D environment created by microspheres, prepared with a fumarate-vinyl acetate copolymer and chitosan. In this system, the environment that the cells feel has similarities to that found by the cells encapsulated in a hydrogel, but the cells have the ability to reorganize their environment since the microspheres are mobile. We evaluated their biocompatibility in vitro using RAW 264.7 macrophages and bone marrow mesenchymal stem cells (BMSCs). The results with RAW 264.7 cells showed good cell viability, without evident signs of cytotoxicity. BMSCs not only proliferate, but also rearrange to grow in clusters, thus highlighting the advantages of microspheres as 3D environments.

Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

BACKGROUND: Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO2 supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential. NEW METHOD: We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber. RESULTS: We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments. COMPARISON WITH EXISTING METHODS: Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO2 gas. Utilizing dry gas supply makes the proposed chamber simple to use. CONCLUSION: Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.

3D spheroid cell cultures and their role in bone regeneration: a systematic review / Cultivos de esferoides 3D y su papel en la regeneración ósea: una revisión sistemática

Abstract Recently, the 3D spheroid cell culture application has been extensively used in the treatment of bone defects. A wide variety of methodologies have been used, which has made the comparison of results complex. Therefore, this systematic review has two aims: (i) to perform an analysis focused on the role of 3D spheroid cell culture in bone regeneration strategies; and (ii) address the main challenges in clinical application. A search of the following keywords "3D cell culture", "spheroid", and "bone regeneration" was carried out in the PubMed, Scopus, and ScienceDirect databases and limited to the years 2010-2020. Studies were included if their primary objective was the behavior of cell aggregates to formed spheroids structures by different 3D cell culture techniques focused on the regeneration of bone tissue. To address the risk of bias for in vitro studies, the United States national toxicology program tool was applied, and descriptive statistics of the data were performed, with the SPSS V.22 program. A total of 16 studies were included, which met the established criteria corresponding to in vitro and in vitro/in vivo studies; most of these studies used stem cells for the 3D cell spheroids. The most often methods used for the 3D formation were low adherence surface and rotational methods, moreover, mesenchymal stem cells were the cell line most frequently used because of their regenerative potential in the field of bone tissue engineering. Although the advances in research on the potential use of 3D spheroids in bone regeneration have made great strides, the constant innovation in cell spheroid formation methodologies means that clinical application remains in the future as strategy for 3D tissue bioprinting.

Resumen Recientemente, la aplicación del cultivo 3D de esferoides se ha utilizado ampliamente en el tratamiento de defectos óseos. La variedad de metodologías para lograr los cultivos 3D de esferoides ha hecho compleja la comparación de resultados. Por tanto, esta revisión sistemática tiene dos objetivos: (i) realizar un análisis centrado en el papel de los cultivos 3D de esferoides en las estrategias de regeneración ósea; y (ii) abordar los principales desafíos en la aplicación clínica. Se realizó una búsqueda de las siguientes palabras clave "cultivo celular 3D", "esferoide" y "regeneración ósea" en las bases de datos PubMed, Scopus y ScienceDirect y se limitó a los años 2010-2020. Se incluyeron los estudios si su principal objetivo era el comportamiento de agregados celulares para generar las estructuras esferoidales desarrollados por diferentes técnicas de cultivo celular 3D enfocadas a la regeneración del tejido óseo. Para abordar el riesgo de sesgo de los estudios in vitro, se aplicó la herramienta del programa nacional de toxicología de Estados Unidos y se realizaron estadísticas descriptivas de los datos, con el programa SPSS V.22. Se incluyeron un total de 16 estudios, que cumplieron con los criterios establecidos correspondientes a estudios in vitro e in vitro/in vivo; la mayoría de estos estudios utilizaron células troncales para generar los esferoides celulares 3D. Los métodos más utilizados para la formación de los esferoides 3D fueron la superficie de baja adherencia y los métodos de rotación, asimismo, la línea celular de células troncales mesenquimales fueron las más utilizadas debido a su gran potencial regenerativo en el campo de la ingeniería de tejidos óseos. Aunque los avances en la investigación sobre el uso potencial de los cultivos celulares de esferoides 3D en la regeneración ósea han logrado grandes avances, la constante innovación en las metodologías de la generación de esferoides 3D deja claro que la aplicación clínica de estos permanecerá en el futuro como estrategia en la bioimpresión tisular.

[Investigation on Cheyletoidea mites breeding in culture environment of Eupolyphaga sinensis and morphologic observation of Eucheyletia reticulate Cunliffe].

OBJECTIVE: To investigate the species of Cheyletoidea mites breeding in the culture environment of Eupolyphaga sinensis and to observe the morphology of Eucheyletia reticulata Cunliffe. METHODS: The soil samples from an E. sinensis farm in northern Anhui were collected. The mites in the soil samples were separated directly under a microscope and the glass specimens were made to observe the morphological feature of the mites under a light microscope, then the mites species were identified and classified based on the morphological characteristics. RESULTS: In the culture soil of E. sinensis, totally 7 kinds of Cheyletoidea mites were isolated, namely Eucheyletia reticulata Cunliffe, Cheyletus eruditus Schrank, Cheyletus malaccensis Oudemans, Cheyletus troussarti Oudemans, Cheyletus aveisor Rohdendorz, Acaropsis sollers Rohdendorz and Cheletomorpha lepidopterorum Shaw. They belonged to genera Eucheyletia, Cheletomorpha, Acaropsis and Cheyletus of Cheyletidae Leach family. The Eucheyletia reticulata Cunliffe was firstly found in the culture environment of E. sinensis, and its gnathosoma was large, the pedipalpal femurs were expanding and there were two strips of comb hair and two smooth bristles on the pedipalpal tarsus, and the back of the body was covered with two pieces of tergum, which were decorated with reticular pattern. The body and foot setae were fan-shape. CONCLUSIONS: There are various of Cheyletoidea mites found in the breeding environment of E. sinensis. These mites are important species for pest control in the culture environments of E. sinensis. Related measures should be taken to prevent the excessive growth of Cheyletoidea mites, so as to avoid the adverse effects on the quality and quantity of E. sinensis.

Soil Physical and Chemical Properties， Microorganisms and Metabolites in Different Culture Environments of Gastrodia elata / 中国实验方剂学杂志

Objective:To study the soil physical and chemical properties， microorganisms， and metabolites in different culture environments of <italic>Gastrodia elata</italic>， so as to provide scientific basis for subsequent cultivation of <italic>G. elata</italic> in multiple environments. Method:The tubersphere soil of <italic>G. elata</italic> cultured in different environments was collected for analyzing the soil nutrients， microbial numbers， and metabolite differences using the agrochemical method， plate-count method， and gas chromatography-time-of-flight mass spectrometry （GC-TOF-MS）-based non-targeted metabonomic approach. Result:The analysis of soil physical and chemical properties revealed the highest soil moisture， pH， available potassium， and available phosphorus in the spinney and the highest electrical conductivity， total nitrogen， alkali-hydrolyzable nitrogen， and organic matter in the pinewood. As demonstrated by the quantitative analysis of soil microorganisms， the cultivable microorganisms were bacteria， actinomycetes， and fungi， with the bacterial population and total microbial biomass in the spinney and the number of fungi and actinomycetes in the barren slope detected to be the largest. The ratio of bacteria to fungi （B/F value） in the pinewood was the highest， while that in the barren slope was the lowest. The results of metabonomic research demonstrated that the compositions and quantities of soil metabolites in the spinney （Z group）， pinewood （S group）， and barren slope （HD group） varied. Through comparisons between S and Z groups， between HD and Z groups， as well as between HD and S groups by orthogonal partial least squares discriminant analysis （OPLS-DA）， 18， 35， and 24 differential metabolites were separately screened out， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis yielded 5， 9， and 13 metabolic pathways. There existed a significant causal relationship of the soil physical and chemical properties and microbial numbers with the metabolites. Conclusion:The soil physical and chemical properties， microbial numbers， and metabolite changes differed significantly in different culture environments of <italic>G. elata</italic>， which were sorted by the suitability in a descending order as follows： spinney > pinewood >barren slope. The soil physical and chemical properties and microbial numbers are the crucial factors driving changes in soil metabolites， suggesting that regulating the soil physical and chemical characteristics and microbial characteristics in the culture environment is an important mechanism for maintaining the <italic>G. elata</italic>-soil-microbial symbiotic system.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos.

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10µM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

Investigation on Cheyletoidea mites breeding in culture environment of Eupolyphaga sinensis and morphologic observation of Eucheyletia reticulata Cunliffe / 中国血吸虫病防治杂志

Objective To investigate the species of Cheyletoidea mites breeding in the culture environment of Eupolyphaga sinensis and to observe the morphology of Eucheyletia reticulata Cunliffe. Methods The soil samples from an E. sinensis farm in northern Anhui were collected. The mites in the soil samples were separated directly under a microscope and the glass speci?mens were made to observe the morphological feature of the mites under a light microscope,then the mites species were identi?fied and classified based on the morphological characteristics. Results In the culture soil of E. sinensis,totally 7 kinds of Chey?letoidea mites were isolated,namely Eucheyletia reticulata Cunliffe,Cheyletus eruditus Schrank,Cheyletus malaccensis Oudemans,Cheyletus troussarti Oudemans,Cheyletus aveisor Rohdendorz,Acaropsis sollers Rohdendorz and Cheletomor?pha lepidopterorum Shaw. They belonged to genera Eucheyletia,Cheletomorpha,Acaropsis and Cheyletus of Cheyletidae Leach family. The Eucheyletia reticulata Cunliffe was firstly found in the culture environment of E. sinensis,and its gnathosoma was large,the pedipalpal femurs were expanding and there were two strips of comb hair and two smooth bristles on the pedipal?pal tarsus,and the back of the body was covered with two pieces of tergum,which were decorated with reticular pattern. The body and foot setae were fan?shape. Conclusions There are various of Cheyletoidea mites found in the breeding environment of E. sinensis. These mites are important species for pest control in the culture environments of E. sinensis. Related measures should be taken to prevent the excessive growth of Cheyletoidea mites,so as to avoid the adverse effects on the quality and quan?tity of E. sinensis.

Gene Expression Analysis of CD34~+ Hematopoietic Stem and Progenitor Cells Grown in Different Culture Environments Using Differential Display / 中国生物工程杂志

Objective: To investigate the changes of gene expression in CD34+ hematopoietic stem and progenitor cells (HSPCs) under different growth environments. Methods: Umbilical cord blood mononuclear cells (UCB MNCs) were cultured in static and stirred systems. After 7 days of culture, CD34+ cells were isolated and total RNA was extracted. Gene expression patterns of CD34+ cells from fresh, static and stirred cultures were compared using differential display (DD). Results: 30 gene fragments displayed differential expression levels based on the conditions of DD. One of differentially expressed genes was identified as RAN, which is a member of oncogene RAS family. This gene may be associated with proliferation of hematopoietic cells. Conclusion: Different growth environments induced differential gene expression patterns of CD34+ HSPCs. These differentially expressed genes would give new insights into optimizing in vitro environments for expanding hematopoietic cells.