One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have 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.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Stimuli-responsive microcarriers and their application in tissue repair: A review of magnetic and electroactive microcarrier

Microcarrier applications have made great advances in tissue engineering in recent years, which can load cells,drugs, and bioactive factors. These microcarriers can be minimally injected into the defect to help reconstruct agood microenvironment for tissue repair. In order to achieve more ideal performance and face more complextissue damage, an increasing amount of effort has been focused on microcarriers that can actively respond toexternal stimuli. These microcarriers have the functions of directional movement, targeted enrichment, materialrelease control, and providing signals conducive to tissue repair. Given the high controllability and designabilityof magnetic and electroactive microcarriers, the research progress of these microcarriers is highlighted in thisreview. Their structure, function and applications, potential tissue repair mechanisms, and challenges are discussed.In summary, through the design with clinical translation ability, meaningful and comprehensiveexperimental characterization, and in-depth study and application of tissue repair mechanisms, stimuliresponsivemicrocarriers have great potential in tissue repair.

Spatiotemporal responsive hydrogel microspheres for the treatment of gastric cancer

The development of tumor drug microcarriers has attracted considerable interest due to their distinctive therapeutic performances.Current attempts tend to elab-orate on the micro/nano-structure design of the microcarriers to achieve multiple drug delivery and spatiotemporal responsive features.Here,the desired hydrogel microspheres are presented with spatiotemporal responsiveness for the treatment of gastric cancer.The microspheres are generated based on inverse opals,their skele-ton is fabricated by biofriendly hyaluronic acid methacrylate(HAMA)and gelatin methacrylate(GelMA),and is thenfilled with a phase-changing hydrogel composed offish gelatin and agarose.Besides,the incorporated black phosphorus quantum dots(BPQDs)within thefilling hydrogel endow the microspheres with outstanding pho-tothermal responsiveness.Two antitumor drugs,sorafenib(SOR)and doxorubicin(DOX),are loaded in the skeleton andfilling hydrogel,respectively.It is found that the drugs show different release profiles upon near-infrared(NIR)irradiation,which exerts distinct performances in a controlled manner.Through both in vitro and in vivo experiments,it is demonstrated that such microspheres can significantly reduce tumor cell viability and enhance the efficiency in treating gastric cancer,indicating a promising stratagem in thefield of drug delivery and tumor therapy.

基于microcarrier 6构建正常免疫小鼠人胃癌移植模型

目的:基于新型3D微载体(microcarrier 6)复合人胃癌MKN45细胞接种于具有正常免疫功能的小鼠,以期建立新型人胃癌正常小鼠转移瘤模型。方法:将60只雄性C57BL/6小鼠按是否将MKN45细胞接种于支架材料随机分为2D组、对照组和3D组;体外构建三维肿瘤细胞培养模型,采用皮下接种法建立小鼠移植瘤模型,观察小鼠成瘤时间、成瘤率、病理学表现等。结果:2D组和对照组均未成瘤,3D组成瘤率达80%,成瘤时间早,移植瘤HE染色和免疫组织化学均符合人胃癌特点。结论:本实验成功基于microcarrier 6复合人胃癌MKN45细胞在正常免疫小鼠中建立人胃癌小鼠移植瘤模型,此模型可以更好地研究和进一步阐明胃癌在免疫功能正常机体中发生、发展机制,同时也为抗癌药物的研发提供了更有价值的动物模型。

正常免疫鼠人原代卵巢癌模型的建立

目的:正常免疫小鼠皮下接种人原代卵巢癌细胞与微载体(microcarrier 6)复合体,建立正常免疫鼠人原代卵巢癌模型。方法:将36只C57BL/6雌鼠按是否将人原代卵巢癌细胞与微载体共同培养随机分为原代卵巢癌细胞组、空载体组和三维原代卵巢癌细胞组。将人原代卵巢癌细胞与microcarrier 6共同培养,于小鼠皮下注射,观察小鼠的成瘤时间、成瘤率和病理学特点。结果:原代卵巢癌细胞组和空载体组均未成瘤,三维原代卵巢细胞组总成瘤率达58%,成瘤快,镜下观察符合人卵巢癌特点。结论:本实验成功建立了正常免疫小鼠人原代卵巢癌模型,该模型能更好地模拟卵巢癌在人体内的发生发展情况,可用于更多抗肿瘤药物研究。

基于microcarrier 6正常免疫鼠人源宫颈癌移植瘤模型的构建及其临床应用意义

目的 基于新型3D微载体(microcarrier 6),复合人原代宫颈癌细胞,皮下接种于具有正常免疫功能的小鼠,以期建立新型宫颈癌人源肿瘤移植瘤动物模型(PDX),并分析正常免疫鼠移植瘤病理学特点。方法 将75只雌性C57BL/6小鼠按移植物不同随机分为三组:细胞对照组、空白对照组和实验组。从新鲜手术标本宫颈癌组织中分离提取原代宫颈癌细胞,并与microcarrier 6共孵育,在体外构建3D微肿瘤模型。将构建的宫颈癌细胞-微载体植入小鼠皮下构建正常免疫小鼠患者来源的宫颈癌移植瘤模型。观察成瘤时间、成瘤率;观察对比肿瘤患者与其对应的移植瘤病理HE染色及应用免疫组化检测P16、CK5/6在各组移植瘤中的表达情况。结果 细胞对照组均没有小鼠成瘤,实验组5次实验,总成瘤率高达88%,成瘤时间10天左右,移植瘤生长速度快,HE染色和免疫组化均符合人宫颈癌特点。结论 本实验基于microcarrier 6复合患者来源的原代宫颈癌细胞在正常免疫小鼠体内成功建立了新型人宫颈癌PDX模型,该模型能更好地揭示宫颈癌的发生发展及转归与机体免疫之间的内在关系,更好地研究宫颈癌在机体中发生、发展机制,为免疫治疗提供了新的更有价值的动物模型。

Alternatives for large-scale production of cultured beef: A review

Cultured beef is a method where stem cells from skeletal muscle of cows are cultured in vitro to gain edible muscle tissue. For large-scale production of cultured beef, the culture technique needs to become more efficient than today＇s 2-dimensional（2D） standard technique that was used to make the first cultured hamburger. Options for efficient large-scale production of stem cells are to culture cells on microcarriers, either in suspension or in a packed bed bioreactor, or to culture aggregated cells in suspension. We discuss the pros and cons of these systems as well as the possibilities to use the systems for tissue culture. Either of the production systems needs to be optimized to achieve an efficient production of cultured beef. It is anticipated that the optimization of large-scale cell culture as performed for other stem cells can be translated into successful protocols for bovine satellite cells resulting in resource and cost efficient cultured beef.

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.

Three-dimensional Expansion:In Suspension Culture of SD Rat's Osteoblasts in a Rotating Wall Vessel Bioreactor

Objective To study large-scale expansion of SD （Sprague-Dawley） rat＇s osteoblasts in suspension culture in a rotating wall vessel bioreactor （RWVB）. Methods The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the rnicrocarriers around 1 dyn/cm^2. The cells were isolated via sequential digestions of neonatal （less than 3 days old） SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope （SEM） for histological examination of the aggregates. Also, the hematoxylin-eosin （HE） staining and alkaline phosphatase （ALP） staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carded out for mineralized nodule formation. Results The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. Conclusions The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional （3D） interactions among cells and is suitable for osteopath expansion in vitro.

Preparation and characterization of chitosan porous microcarriers for hepatocyte culture

BACKGROUND: The bioartificial liver (BAL) is considered a possible alternative method for treating liver failure. The core of the BAL system is culturing liver cells in vitro with high density and activity. Microcarrier culture is a mode of high-density culture. We set out to prepare a novel porous microcarrier to improve the activity of liver cells in vitro. METHODS: Chitosan was used to prepare a novel porous spherical microcarrier with interconnected structure. The chitosan porous microcarriers (CPMs) were modified with gelatin to improve their biocompatibility. CPMs were co-cultured with liver cells, HL-7702 (L-02), to evaluate their effect on cell culture. RESULTS: The average size of the CPMs was about 400 μm in diameter and their apertures were less than 30 μm. The pores of the microcarrier were interconnected. After fixation by sodium tripolyphosphate, the structure of the first freeze-dried CPMs was stable. To further improve the biocompatibility, the surface of CPMs was modified with gelatin through chemical crosslinking (GM-CPMs). Comparing the proliferation curves of L-02 cells cultured on simple CPMs, GM-CPMs and tissue culture polystyrene (TCPS, a mode of planar cell culture), the proliferation rates were similar in the first 5 days and the cells proliferated until day 8 in culture with microcarriers. The OD value of liver cells cultured on GM-CPMs was 1.97-fold higher than that on TCPS culture at day 8. Levels of urea and albumin in supernatants of cells cultured on GM-CPMs increased steadily for 8 days, and were clearly higher than those of cells cultured on TCPS (P<0.05).CONCLUSIONS: The novel CPMs were promising microcarriers for hepatocyte culture and the GM-CPM seemed better. Porous microcarrier culture was beneficial for hepatocyte function and activity.

Increased production of recombinant prourokinase with porous microcarrier cell culture by periodic pressure oscillation in a stirred tank reactor

An rCHO cell line expressing recombinant human prourokinase （pro-UK） at the level of 5μg/ 10^6cells/d was cultivated on Cytopore cellulose porous microcarriers in a 7.5L Biostat CT stirred tank reactor. A periodic pressure oscillation of 0.04 MPa and 0.04 Hz was adopted to introduce a physical stimulus on the rCHO cells and to improve mass transfer characteristic between cells and medium in the process of porous microcarrier CHO cell culture. Compared to constant pressure culture, the oscillation culture didn＇t influence specific cell growth rate significantly, but could enhance the pro-UK specific production by 10% - 40%, and reduce production of lactate by 10% - 30%. In the perfusion culture of recombinant CHO cell with serum-free medium for 67 days, cell density could reach 2.64×10^7/ml, the maximal prourokinase concentration in harvested supernatant was about 118mg/L, a total of 21.1 grams of prourokinase was produced in 313 liters of supernatant. In conclusion, the perfusion cell culture with periodic pressure oscillation can enhance the production of recombinant protein and increase the reactor specific productivity.

HIGH DENSITY CULTIVATION OF A RECOMBINANT CD－1 CELL LINE PRODUCING PROUROKINASE USING A BIOSILON MICROCARRIER CULTURE SYSTEM

CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density (over1×107cells/ml). Prourokinase was stably secreted at about 180 IU/ 1e6 cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reatthch and proliferate on fresh microcarriers. This makes it very easy to scale up preduction. The microcarriers could be reused several times without affecting adhesion. proliferation and prourokinase secretion. With CMPECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1×105 IU/mg of protein. The purification factor was about 600 fold, and approxiamately 90 % of the biological activity was recovered.

HIGH DENSITY CULTIVATION OF GENETICALLY－ENGINEERED CHO CELL LINES WITH MICROCARRIER CULTURE SYSTEMS

Genetically-engineered CHO cell lines, rβ- 13 and CLF-8B2, were cultivated with the MC- 1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml. the cell density could reach 4 to 5 × 106 cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method. i. e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system. we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0. 5 to 3working volumes. Prourokinase was stably secreted.

Research Progress on the Large-scale Culture Technology of Mammalian Cells

The culture of mammalian cells is closely related to the development of biotechnology, which has been used extensively in the research and application fields of biology and medical science. In this article, various factors affecting cell cultivation and the application of microcarrier and bioreactor on large-scale culture of mammalian ceils were reviewed.

Cultivating human liver cell line (CL-1) on microcarriers as biomaterial of bioartificial liver

objective: To cultivate human liver cell line (CL-1) on microcarriers and study the synthetic and transformational function of this culture system. Methods:CL-1 were cultivated on Cytodex-3 microcarriers. The cell growth was kinetically inspected with light microscope and scanning electronic microscope on the lst, 3rd, 5th, 7th, 9th day, and the amount of diazepam transformation and albumin synthesis were deter mined at the same time. Results:On 7th day after inoculating, the CL-1 cell density could reach 2. 16 ×106/ ml ; the amount of diazepam trans formation was 619 μg and albumin synthesis 78. 23 μg. Conclusion:CL-1 can be cultivated to a high density on microcarriers and has hepatic specific biotransformation and biosynthesis functions. So the culture system may be further studied for being used as the biomaterial of bioartificial liver.

The Growth Study of Vero Cells in Different Type of Microcarrier

The fact of microcarrier (MC) culture introduces new possibilities and makes possible the practical high-yield culture of anchorage-dependent cells has generated a considerable focus in this study. The objective of this research was to study the comparison of Vero cell growth on different types of commercial microcarriers;Cytodex-1, Cytodex-3, Hil-lex? II and Plastic Plus in spinner vessel and two liters bioreactor cultured for 96 hours. Biological performance of the microcarrier in RPMI media showed the preference of Vero cell grew on Cytodex 3 microcarriers with highest maximum viable cell number (2.4 × 105 cells/ml) followed by Cytodex 1, Hillex and Plustic Plus. Vero cell on Cyto-dex-3 data in spinner flask was compared in bioreactor and result showed higher viable cell number in biorector. Thus, this dextran-crosslink gelatin microcarrier (Cytodex 3) provided the best surface for cell attachment and fast proliferation. At the end of this cell growth improvement will be used for virus transfection producing a vaccine in bioreactor.

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.

THE PREPARATION OF FRUCTOSE-MODIFIED CHITOSAN MICROCARRIERS AND CULTURE OF PRIMARY RAT HEPATOCYTES

The fructose modified chitosan microcarries (CMs) were prepared by the reaction of glutaraldehyde with fructose-modified chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Morphology of rat hepatocytes cultured on CMs was observed using phase contrast microscope and scanning electron microscope, and the metabolic activities were measured. Rat hepatocytes cultured on CMs retained the spherical shape as they have in vivo and had high metabolic activities. Fructose can enhance the metabolic activity of hepatocytes and the modified CMs are promising scaffold for hepatocytes attachment.

PREPARATION AND THE CULTURE OF LO2 CELLS ON PVA-BASED MICROCARRIERS

Using polyvinyl alcohol (PVA) as raw material and vacuum pump oil as oil phase medium, PVA-based microcarriers were prepared by suspension method. The diameters of the beads were 100-180μm. LO2 cells were cultured on PVA-based microcarriers and cytodexIII microcarriers. Morphology, attachment and growth rate of LO2 cells were studied.

Constructing thermoresponsive PNIPAM-based microcarriers for cell culture and enzyme-free cell harvesting

The development of large-scale cell cultivation and non-invasive cell harvesting is highly desired in various fields,including biological regeneration and pharmaceutical research.When using traditional microcarriers for cell culture,trypsinization is often necessary during cell collection,leading to partial cells damage.In this work,we developed a thermoresponsive glass microcarrier modified with poly(γ-propargyl-L-glutamate)(PPLG)and poly(N-isopropylacrylamide)(PNIPAM).We utilized these microcarriers for three-dimensional cell culture and enzyme-free cell harvesting,and the results indicated that the prepared microcarriers exhibited excellent non-invasive cell culture performance.