Caspase-9 suppresses metastatic behavior of MDA-MB-231 cells in an adaptive organoid model.

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells.

Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

OBJECTIVES: The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. METHODS: Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24 h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). RESULTS: Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. CONCLUSIONS: The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. CLINICAL SIGNIFICANCE: Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Colon cancer inhibitory properties of Caulerpa lentillifera polysaccharide and its molecular mechanisms based on three-dimensional cell culture model.

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333-8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.

Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

Generating and Characterizing Adipose Spheroids from Adipose-Derived Stromal/Stem Cells.

Advances in technology and automation over the past several decades have made it feasible to perform high-throughput compound screening with cell spheroids, a valuable approach for drug discovery. It is entirely feasible to generate multiple 384-well plates containing adipose spheroids from cryopreserved, single-donor, adipose stem cells, thus incorporating genetic diversity into the discovery stages of research. In this protocol, we describe our method for isolating primary human adipose stem cells and synthesizing cell spheroids comprised of mature adipocytes and stromal cells. Also included are representative outcome measurements useful for characterizing adipocyte metabolism and health. Wherever possible, we describe technologies that can be used to automate characterization and increase throughput.

Functional Assays in 3D ObaCell® Fat-on-a-Chip Cultures: Lipolysis and Glucose Uptake Assays.

Advancements in three-dimensional in vitro cultures pose a need for modification of established two-dimensional culture functional assay methods. Application of three-dimensional in vitro models in drug screening and target validation, specifically in the development of compounds targeting adipose metabolic activity, requires optimization of current glucose uptake and lipolysis assay protocols to effectively measure adipocyte function in a three-dimensional platform. This chapter describes the establishment of three-dimensional cultures using Obatala Sciences' human-derived hydrogel, maintenance and treatment of the cultures, and evaluation of compound response via lipolysis and glucose uptake assays.

A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Reflecting the human lip in vitro: Cleft lip skin and mucosa keratinocytes keep their identities.

OBJECTIVES: Cell models have shown great promise as tools for research, potentially providing intriguing alternatives to animal models. However, the original tissue characteristics must be maintained in culture, a fact that is often assumed, but seldom assessed. We aimed to follow the retention of the original tissue identities of cleft lip-derived skin and mucosa keratinocytes in vitro. METHODS: Cleft lip-derived keratinocytes were isolated from discarded tissue along the cleft margins during cheiloplasty. Cell identities were assessed by immunohistochemistry and quantitative real-time PCR for tissue-specific markers and compared with native lip tissue. Moreover, keratinocytes were regularly analyzed for the retention of the original tissue characteristics by the aforementioned methods as well as by differentiation assays. RESULTS: The various anatomical zones of the human lip could be distinguished using a panel of differentiation and functional-based markers. Using these markers, retention of the original tissue identities could be followed and confirmed in the corresponding primary keratinocytes in culture. CONCLUSIONS: Our findings promote patient-derived cells retaining their original identities as astonishing and clinically relevant in vitro tools. Such cells allow a better molecular understanding of various lip-associated pathologies as well as their modeling in vitro, including but not restricted to orofacial clefts.

Insights on Three Dimensional Organoid Studies for Stem Cell Therapy in Regenerative Medicine.

Regenerative medicine has developed as a promising discipline that utilizes stem cells to address limitations in traditional therapies, using innovative techniques to restore and repair damaged organs and tissues. One such technique is the generation of three-dimensional (3D) organoids in stem cell therapy. Organoids are 3D constructs that resemble specific organs' structural and functional characteristics and are generated from stem cells or tissue-specific progenitor cells. The use of 3D organoids is advantageous in comparison to traditional two-dimensional (2D) cell culture by bridging the gap between in vivo and in vitro research. This review aims to provide an overview of the advancements made towards regenerative medicine using stem cells to generate organoids, explore the techniques used in generating 3D organoids and their applications and finally elucidate the challenges and future directions in regenerative medicine using 3D organoids.

The mannose-binding protein from Agaricus bisporus inhibits the growth of MDA-MB-231 spheroids.

A mannose-binding protein from the mushroom Agaricus bisporus (Abmb) inhibits the growth of MDA-MB-231 cells, which is of an aggressive breast cancer subtype. This ability was observed in a monolayer cell (2D) culture setup, which often is unable to capture changes in cell morphology, polarity and division. That shortcoming may overestimate Abmb potency for its development as a pharmaceutical agent and its use in a therapy. Hence, Abmb's inhibition to the cell growth was performed in the 3D cell (spheroid) culture, which is more representative to the situation in vivo. The result showed that, although the presence of Abmb at ~14.7 µM already disrupted the MDA-MB-231 cell morphology in the 2D culture, its presence at ~16.5 µM only ceased the growth of the MDA-MB-231 spheroid. Further, Abmb is unique because structurally it belongs to the R-type lectin (RTL) family; most of mannose-binding protein is of the C-type lectin (CTL). As the natural ligand of Abmb is unknown thus the mechanism of action is unclear, Abmb effect on the cancer cells was assessed via observation of the altered expression of genes involved in the Wnt/ß-catenin signalling, which is one of the canonical pathways in the proliferation of cancer cells. The results suggested that Abmb did not alter the pathway upon exerting its anti-proliferative activity to the MDA-MB-231 cells.

Mesenchymal stem cells lose the senescent phenotype under 3D cultivation.

BACKGROUND: Three-dimensional (3D) cell culture is widely used in various fields of cell biology. In comparison to conventional two-dimensional (2D) cell culture, 3D cell culture facilitates a more accurate replication of the in vivo microenvironment, which is essential for obtaining more relevant results. The application of 3D cell culture techniques in regenerative medicine, particularly in mesenchymal stem cell (MSC)-based research, has been extensively studied. Many of these studies focus on the enhanced paracrine activity of MSCs cultured in 3D environments. However, few focus on the cellular processes that occur during 3D cultivation. METHODS: In this work, we studied the changes occurring within 3D-cultured MSCs (3D-MSCs). Specifically, we examined the expression of numerous senescent-associated markers, the actin cytoskeleton structure, the architecture of the Golgi apparatus and the localization of mTOR, one of the main positive regulators of replicative senescence. In addition, we assessed whether the selective elimination of senescent cells occurs upon 3D culturing by using cell sorting based on autofluorescence. RESULTS: Our findings indicate that 3D-MSCs were able to lose replicative senescence markers under 3D cell culture conditions. We observed changes in actin cytoskeleton structure, Golgi apparatus architecture and revealed that 3D cultivation leads to the nuclear localization of mTOR, resulting in a decrease in its active cytoplasmic form. Additionally, our findings provide evidence that 3D cell culture promotes the phenotypic reversion of senescent cell phenotype rather than their removal from the bulk population. CONCLUSION: These novel insights into the biology of 3D-MSCs can be applied to research in regenerative medicine to overcome replicative senescence and MSC heterogeneity as they often pose significant concerns regarding safety and effectiveness for therapeutic purposes.

A Simple and Fast Method for the Formation and Downstream Processing of Cancer-Cell-Derived 3D Spheroids: An Example Using Nicotine-Treated A549 Lung Cancer 3D Spheres.

Although 2D in vitro cancer cell cultures have been used for decades as a first line-of-research tool to investigate antitumoral drugs and treatments, their use presents many drawbacks, including the poor resemblance of such cultures to the characteristics of in vivo tumors. To mitigate these drawbacks, 3D culture models have emerged as a more representative alternative. Cancer cells cultured as 3D structures have the advantage of resembling solid tumors in their architecture and in their resistance to chemotherapeutic drugs, in part because of restrained drug penetration. Additionally, these 3D structures create a more physiological environment for the study of immune cell invasion and migration, comparable to solid tumors. In this paper, we describe a fast and cost-effective step-by-step protocol for the generation of 3D spheres using ultra-low-attachment (ULA) multiwell plates, which can be incorporated into the normal workflow of any laboratory. Using this protocol, spheroids of different human cancer cell lines can be obtained and can then be characterized on the basis of their morphology, viability, and expression of specific markers.

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

The development and characterization of in vivo-like three-dimensional models of bronchial epithelial cell lines.

In vitro models of differentiated respiratory epithelium that allow high-throughput screening are an important tool to explore new therapeutics for chronic respiratory diseases. In the present study, we developed in vivo-like three-dimensional (3-D) models of bronchial epithelial cell lines that are commonly used to study chronic lung disease (16HBE14o-, CFBE41o- and CFBE41o- 6.2 WT-CFTR). To this end, cells were cultured on porous microcarrier beads in the rotating wall vessel (RWV) bioreactor, an optimized suspension culture method that allows higher throughput experimentation than other physiologically relevant models. Cell differentiation was compared to conventional two-dimensional (2-D) monolayer cultures and to the current gold standard in the respiratory field, i.e. air-liquid interface (ALI) cultures. Cellular differentiation was assessed in the three model systems by evaluating the expression and localization of markers that reflect the formation of tight junctions (zonula occludens 1), cell polarity (intercellular adhesion molecule 1 at the apical side and collagen IV expression at the basal cell side), multicellular complexity (acetylated α-tubulin for ciliated cells, CC10 for club cells, keratin-5 for basal cells) and mucus production (MUC5AC) through immunostaining and confocal laser scanning microscopy. Results were validated using Western Blot analysis. We found that tight junctions were expressed in 2-D monolayers, ALI cultures and 3-D models for all three cell lines. All tested bronchial epithelial cell lines showed polarization in ALI and 3-D cultures, but not in 2-D monolayers. Mucus secreting goblet-like cells were present in ALI and 3-D cultures of CFBE41o- and CFBE41o- 6.2 WT-CFTR cells, but not in 16HBE14o- cells. For all cell lines, there were no ciliated cells, basal cells, or club cells found in any of the model systems. In conclusion, we developed RWV-derived 3-D models of commonly used bronchial epithelial cell lines and showed that these models are a valuable alternative to ALI cultures, as they recapitulate similar key aspects of the in vivo parental tissue.

A Novel NCI-H69AR Drug-Resistant Small-Cell Lung Cancer Mini-Tumor Model for Anti-Cancer Treatment Screening.

Small-cell lung cancer is a fast-growing carcinoma with a poor prognosis and a high level of relapse due to multi-drug resistance (MDR). Genetic mutations that lead to the overexpression of efflux transporter proteins can contribute to MDR. In vitro cancer models play a tremendous role in chemotherapy development and the screening of possible anti-cancer molecules. Low-cost and simple in vitro models are normally used. Traditional two-dimensional (2D) models have numerous shortcomings when considering the physiological resemblance of an in vivo setting. Three-dimensional (3D) models aim to bridge the gap between conventional 2D models and the in vivo setting. Some of the advantages of functional 3D spheroids include better representation of the in vivo physiology and tumor characteristics when compared to traditional 2D cultures. During this study, an NCI-H69AR drug-resistant mini-tumor model (MRP1 hyperexpressive) was developed by making use of a rotating clinostat bioreactor system (ClinoStar®; CelVivo ApS, Odense, Denmark). Spheroid growth and viability were assessed over a 25-day period to determine the ideal experimental period with mature and metabolically stable constructs. The applicability of this model for anti-cancer research was evaluated through treatment with irinotecan, paclitaxel and cisplatin for 96 h, followed by a 96 h recovery period. Parameters measured included planar surface area measurements, estimated glucose consumption, soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase levels, histology and efflux transporter gene expression. The established functional spheroid model proved viable and stable during the treatment period, with retained relative hyperexpression of the MRP1 efflux transporter gene but increased expression of the P-gp transporter gene compared to the cells cultured in 2D. As expected, treatment with the abovementioned anti-cancer drugs at clinical doses (100 mg/m2 irinotecan, 80 mg/m2 paclitaxel and 75 mg/m2 cisplatin) had minimal impact on the drug-resistant mini-tumors, and the functional spheroid models were able to recover following the removal of treatment.

Considering the Value of 3D Cultures for Enhancing the Understanding of Adhesion, Proliferation, and Osteogenesis on Titanium Dental Implants.

BACKGROUND: Individuals with pathologic conditions and restorative deficiencies might benefit from a combinatorial approach encompassing stem cells and dental implants; however, due to the various surface textures and coatings, the influence of titanium dental implants on cells exhibits extensive, wide variations. Three-dimensional (3D) cultures of stem cells on whole dental implants are superior in testing implant properties and were used to examine their capabilities thoroughly. MATERIALS AND METHODS: The surface micro-topography of five titanium dental implants manufactured by sandblasting with titanium, aluminum, corundum, or laser sintered and laser machined was compared in this study. After characterization, including particle size distribution and roughness, the adhesion, proliferation, and viability of adipose-derived stem cells (ADSCs) cultured on the whole-body implants were tested at three time points (one to seven days). Finally, the capacity of the implant to induce ADSCs' spontaneous osteoblastic differentiation was examined at the same time points, assessing the gene expression of collagen type 1 (coll-I), osteonectin (osn), alkaline phosphatase (alp), and osteocalcin (osc). RESULTS: Laser-treated (Laser Mach and Laser Sint) implants exhibited the highest adhesion degree; however, limited proliferation was observed, except for Laser Sint implants, while viability differences were seen throughout the three time points, except for Ti Blast implants. Sandblasted surfaces (Al Blast, Cor Blast, and Ti Blast) outpaced the laser-treated ones, inducing higher amounts of coll-I, osn, and alp, but not osc. Among the sandblasted surfaces, Ti Blast showed moderate roughness and the highest superficial texture density, favoring the most significant spontaneous differentiation relative to all the other implant surfaces. CONCLUSIONS: The results indicate that 3D cultures of stem cells on whole-body titanium dental implants is a practical and physiologically appropriate way to test the biological characteristics of the implants, revealing peculiar differences in ADSCs' adhesion, proliferation, and activity toward osteogenic commitment in the absence of specific osteoinductive cues. In addition, the 3D method would allow researchers to test various implant surfaces more thoroughly. Integrating with preconditioned stem cells would inspire a more substantial combinatorial approach to promote a quicker recovery for patients with restorative impairments.

[Progress on three-dimensional cell culture technology and their application].

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.

A Three-Dimensional Primary Cortical Culture System Compatible with Transgenic Disease Models, Virally Mediated Fluorescence, and Live Microscopy.

In vitro cell culture models can offer high-resolution and high-throughput experimentation of cellular behaviors. However, in vitro culture approaches often fail to fully recapitulate complex cell processes involving synergistic interactions between heterogeneous neural cell populations and the surrounding neural microenvironment. Here, we describe the formation of a three-dimensional primary cortical cell culture system compatible with live confocal microscopy.