Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish

Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar W. Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2013-4997

A novel in vitro model of human implantation: an endometrium-like three-dimensional (3D) culture system for attachment/invasion of trophoblast-like cells

BackgroundExosomes derived from mesenchymal stem cells (MSC-exos) have been demonstrated with great potential in the treatment of multiple human diseases including acute kidney injury (AKI) by virtue of their intrinsic cargoes. However, there are major challenges of low yield and the lack of an established biomanufacturing platform to efficiently produce MSC-exos, thereby limiting their therapeutic application. Here, we aimed to establish a novel strategy to produce MSC-exos with a hollow fiber bioreactor-based three-dimensional (3D) culture system and evaluate the therapeutic efficacy of 3D-exosomes (3D-exos) on AKI.MethodsMesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in two-dimensional (2D) flasks. 2 × 108 MSCs were inoculated into the hollow fiber bioreactor for 3D culture. The culture supernatants were collected every 1 or 2 days for isolating exosomes. Exosomes from 2D (2D-exos) and 3D cultures were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis of exosome markers. The yield of exosomes from 2 × 108 MSCs seeded in 2D and 3D culture system was compared, based on protein quantification. The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro.Results3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers of 3D-exos when compared to those of 2D-exos. Compared with conventional 2D culture, the 3D culture system increased total exosome production up to 19.4-fold. 3D-exos were more concentrated in the harvested supernatants (15.5-fold) than 2D-exos, which led to a higher exosome collection efficiency of 3D culture system. In vivo, both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function, attenuated pathological changes of renal tubules, reduced inflammatory factors, and repressed T cell and macrophage infiltration. Impressively, 3D-exos were more effective than 2D-exos. Moreover, 3D-exos were taken up by tubular epithelial cells (TECs) with improved efficiency, thereby exhibiting superior anti-inflammatory effect and improved viability of TECs in vitro.ConclusionsIn summary, our findings demonstrate that the hollow fiber 3D culture system provides an efficient strategy for the continuous production of MSC-exos which has enhanced therapeutic potential for cisplatin-induced AKI.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.

To investigate the effect of insulin-like growth factor-I (IGF-I) on the proliferation and osteogenesis of human periodontal ligament stem cells (hPDLC) under three-dimensional (3D) culture system. Human periodontal cells were isolated from the ligament of surgically extracted human teeth, and through the limiting dilution assay, got mono-clone of the cell, hPDLCs were isolated from MesenPRO RS medium. Rotary cell culture system (RCCS) was enrolled to set 3D environment. Control group and experiment groups were assigned according to the concentration of IGF-I. There were 5 level of experiment groups (0.1, 1, 10, 50, 100 µg/L). Proliferation was tested with methyl thiazolyl tetrazolium (MTT), and alkine phosphatase (ALP) level was assayed by spectrophotometer to analyze the osteogenesis of hPDLCs. Gene expression of ostetocalcin (OCN) and type I collagen (Col I) were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). In 3D culture system, the effect of IGF-I on cell proliferation was significantly different between control group and experiment groups (P < 0.05), and there showed significant differences between the group of 0.1 µg/L (0.219 ± 0.021) IGF-I and the groups of 50, 100 µg/L (0.287 ± 0.011, 0.293 ± 0.012). However, there showed no significant differences among other groups. Significant differences of ALP activity were observed between the control group and experiment groups, and between the groups of 1, 10 µg/L (0.304 ± 0.020, 0.310 ± 0.013) and that of 50, 100 µg/L (0.347 ± 0.011, 0.344 ± 0.010) (P < 0.05). While no significant differences were detected between the group of 1 µg/L and that of 10 µg/L, nor between the group of 50 µg/L and that of 100 µg/L. Expressions of Col I and OCN in mRNA and protein level both showed dose-dependent increase. In 3D culture system, in the scale of 0.1 - 100 µg/L, the effect of IGF-I on the proliferation of hPDLCs increased dose-dependently. 100 µg/L IGF-I promotes osteogenesis of the cells significantly.

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1±0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro.

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has beenused for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3Dculture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levelsof secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured inisolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. Inisolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did notaffect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, themRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphologybetween them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonalresponsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

Background: Three-dimensional (3D) culture systems use polymer particles with a bone marrow stroma cell feeder layer to reproduce a biostructural hematopoiesis state more effectively than in conventional two-dimensional (2D) culture methods. The 3D culture maintains normal hematopoiesis, resulting in prolongation of hematopoietic stem cell proliferation and differentiation, while the bone marrow stromal cells in the culture alter the growth of leukemic cells and protect them from anticancer agents. However, the effect of stromal cells on hematopoietic stem cell proliferation and differentiation and neoplastic cells, including leukemia, in 3D culture is still a point of contention. Methods: We assessed the mechanism of two different bone-marrow-derived stromal cells (i.e., MS-5 and Tst-4) with different characteristics by using a feeder layer in the 3D culture to compare their supportive action on leukemic cells, focusing on the role of 3D cultures constructed with bone marrow stromal cells in leukemic cell growth. Multiple myeloma cells are strongly related to stromal cells in their proliferation; hence, cloned MM1.S cells derived from multiple myeloma were cocultured in 3D, and their cell growth was examined. We also examined the effect of the antineoplastic agent bortezomib, a proteasome inhibitor, in the 3D culture system with a different stromal cell feeder. Results and Conclusions: When MM1.S myeloma cells were cultured with MS-5 stroma in 3D conditions, cell growth was found to be slow compared with that in 2D culture, as well as with those in both the 2D and 3D cocultures with Tst-4 stroma. Additionally, the MS-5 cells in the 3D culture protected the MM1.S cells from the cytocidal effect of the bortezomib treatment. Different MM1.S cell kinetics were observed depending on the stromal cells used, suggesting their inherent and complicated characteristics.

The tumor microenvironment contributes significantly to tumor initiation, progression, and resistance to chemotherapy. Much of our understanding of the tumor and its microenvironment is developed using various methods of cell culture. Throughout the last two decades, research has increasingly shown that 3D cell culture systems can remarkably recapitulate the complexity of tumor architecture and physiology compared to traditional 2D models. Unlike the flat culture system, these novel models enabled more cell-cell and cell-extracellular matrix interactions. By mimicking in vivo microenvironment, 3D culture systems promise to become accurate tools ready to be used in diagnosis, drug screening, and personalized medicine. In this review, we discussed the importance of 3D culture in simulating the tumor microenvironment and focused on the effects of cancer cell-microenvironment interactions on cancer behavior, resistance, proliferation, and metastasis. Finally, we assessed the role of 3D cell culture systems in the contexts of drug screening. 2D culture system is used to study cancer cell growth, progression, behavior, and drug response. It provides contact between cells and supports paracrine crosstalk between host cells and cancer cells. However, this system fails to simulate the architecture and the physiological aspects of in vivo tumor microenvironment due to the absence of cell-cell/ cell-ECM interactions as well as unlimited access to O2 and nutrients, and the absence of tumor heterogeneity. Recently advanced research has led researchers to generate 3D culture system that can better recapitulate the in vivo environment by providing hypoxic medium, facilitating cell-cell and cell-ECM, interactions, and recapitulating heterogeneity of the tumor. Several approaches are used to maintain and expand cancer cells in 3D culture systems such as tumor spheroids (cell aggregate that mimics the in vivo growth of tumor cells), scaffold-based approaches, bioreactors, microfluidic derives, and organoids. 3D systems are currently used for disease modeling and pre-clinical drug testing.

[BACKGROUND] In vitro chemosensitivity tests are not always consistent to clinical results (Hutchinston L et al., Nat Rev Clin Oncol. 2011), partly because of lacking extracellular matrix-cell interactions in 2-dimensional (2D) cell culture system (Antoni D et al., Int J Mol Sci. 2015). Three-dimensional cell culture (3D) system maintains cells with a higher degree of structural complexity and homeostasis, analogous to tissues and organs compared with 2D cell culture system. Thus, 3D cell culture system will be expected for alteration of the animal experiment in chemosensitivity test (Bokhari M et al., J Anat. 2007). Cytotoxic agents are crucial for treating triple negative breast cancer (TNBC).[AIM] To clarify differences of drug sensitivities between 2D and 3D culture in TNBC cell lines, we compared IC50s for Cisplatin (CDDP), Docetaxel (DTX), Epirubicin (EPI) and Bromodomain inhibitor (JQ1). [METHODS] Thirteen TNBC cell lines were examined; HCC1599, HCC1937, HCC38, MDA-MB-468, HCC1806, HCC70, MDA-MB-436, MDA-MB-231, Hs578T, BT549, MDA-MB-453, BT20, and HCC1395. For 2D culture, cells were plated on general 96 well cell culture plate for 24 hours. For 3D culture, cells were plated on the PrimeSurface® 96 well plate (SUMITOMO BAKELITE) for 72 hours. Then, those were incubated in media including CDDP, DTX, EPI or JQ1 for 72 hours. The cell viability was quantified using CellTiter-Glo 2.0 and CellTiter-Glo 3D cell viability Assay (Promega), respectively. [RESULTS] For CDDP, IC50s were highly correlated between 2D and 3D culture (r=0.928) . The mean of IC50 value showed higher in 3D culture (27.67±8.40μM) than 2D culture (12.86 ± 17.23μM). For EPI, the correlation was r=0.593. The mean value showed higher in 3D culture (1.41±1.30μM) than 2D culture (0.62±0.72μM). For DTX and JQ1, the mean values were 1.78 ± 2.05nM and 4.31 ±4.72μM in 2D culture, respectively. However, IC50 failed to be obtained in several TNBC cell lines (HCC1937, HCC38, HCC1806, Hs578T and HCC1395 for JQ1 and MDA-MB-436, MDA-MB-453 and BT20 for DTX) in 3D culture. [CONCLUSION] IC50 value was higher in 3D culture than in 2D culture for CDDP and EPI. The correlation of IC50s between 2D and 3D depended on drugs with the highest in CDDP. We are under investigation on reasons why sensitivity tests for DTX and JQ1 failed in 3D culture system. Citation Format: Masako Muguruma, Saeko Teraoka, Kana Miyahara, Ai Ueda, Takahiko Kawate, Takashi Ishikawa. Differences of drug sensitivities between 2-dimensional and 3-dimensional culture systems in triple negative breast cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 326.

Neutrophils (Neu) migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis, including the intestinal epithelia injuries and immune responses. Although their important roles in these diseases, they are challenging to study due to their short life span and the inability to cryopreserve or expand them in vitro. Moreover, the standard cell culturing on plastic plates (two-dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. In this study, we developed a new three-dimensional (3D) culture system for human and mouse peripheral blood Neu, which is made of hydrogel. The Neu showed much better cell integrity and less cell debris in the 3D culture system compared to that in 2D culture system. Moreover, the 3D culture system was more suitable for the observation of neutrophil extracellular traps (NETs) stimulated by the classical stimulation phorbol ester (PMA), and other damage associated molecular patterns (DAMPs) such as Lipopolysaccharide (LPS)/ATP, interleukin-1 β (IL-1β) and tumor necrosis factor α (TNFα) than the 2D culture system. Moreover, NETs phenomenon in 3D culture system is similar to that in vivo. In addition, the 3D culture system was evaluated to co-culturing Neu and other parenchymal cells, such as colon mucosal epithelial cell lines. In conclusion, the 3D culture system could maintain better properties of Neu than that in 2D culture system and it may reduce the gap between in vitro an in vivo experimentations.

Leukemia Bone Marrow Primary Cells Long-Term Culture in a Biomimetic Hematopoietic Niche.

Drug-induced liver injury (DILI) is the major reason for failures in drug development and withdrawal of approved drugs from the market. Two-dimensional cultures of hepatocytes often fail to reliably predict DILI: hepatoma cell lines such as HepG2 do not reflect important primary-like hepatic properties and primary human hepatocytes (pHHs) dedifferentiate quickly in vitro and are, therefore, not suitable for long-term toxicity studies. More predictive liver in vitro models are urgently required in drug development and compound safety evaluation. This review discusses available human hepatic cell types for in vitro toxicology analysis and their usage in established and emerging three-dimensional (3D) culture systems. Generally, 3D cultures maintain or improve primary hepatic functions (including expression of drug-metabolizing enzymes) of different liver cells for several weeks of culture, thus allowing long-term and repeated-dose toxicity studies. Spheroid cultures of pHHs have been comprehensively tested, but also other cell types such as HepaRG benefit from 3D culture systems. Emerging 3D culture techniques include usage of induced pluripotent stem-cell-derived hepatocytes and primary-like upcyte cells, as well as advanced culture techniques such as microfluidic liver-on-a-chip models. In-depth characterization of existing and emerging 3D hepatocyte technologies is indispensable for successful implementation of such systems in toxicological analysis.

Three dimensional (3D) models mimic the features of native tissue environment. Thus, morphology and signalling of cells from 3D culture are often more physiological than routine two dimensional (2D) cell culture. It is also known that the cell-secreted products have paracrine effect on other cells growth. In this experimental study, we optimised the nasal fibroblast culture on a 3D cell culture system and studied the effects of secretome from the 3D culture (3DCM) onto fibroblast growth and cytoprotection. Nasal fibroblast was isolated from human nasal turbinates. The suitable microcarrier was selected by culturing the fibroblasts in passage 3 on various types PolyGEM™ polystyrene microcarriers. Then, the cells were cultured on selected microcarrier using a 3D culture system and the conditioned medium (CM) was collected. 3DCM were supplemented to fibroblasts to study for attachment, proliferation, and cytoprotective effects against cytotoxicity of Centella asiatica. Bicinchonic Acid Assay (BCA) was performed to quantify protein amount in CMs. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for preliminary profiling and comparison of 2DCM and 3DCM protein profile. Our study showed that the 3DCM did not significantly enhance cell attachment and proliferation. The secretome of both 2DCM and 3DCM found to have significant cytoprotective effect onto nasal fibroblast against cytotoxicity of C.asiatica extract. 3DCM had higher protein concentration than 2DCM. SDS-PAGE showed three exclusive proteins in 3DCM and four exclusive proteins in 2DCM. Future study should be conducted on utility of nasal fibroblast secretome on cytoprotection against harmful agents in environment and cytotoxicity of natural products.