Targeted immunotherapy for glioblastoma involving whole tumor-derived autologous cells in the upfront setting after craniotomy.

PURPOSE: To date, immunotherapeutic approaches in glioblastoma (GBM) have had limited clinical efficacy as compared to other solid tumors. Here we explore autologous cell treatments that have the potential to circumvent treatment resistance to immunotherapy for GBM. METHODS: We performed literature review and assessed clinical outcomes in phase 1 safety trials as well as phase 2 and 3 autologously-derived vaccines for the treatment of newly-diagnosed GBM. In one recent review of over 3,000 neuro-oncology phase 2 and phase 3 clinical trials, most trials were nonblinded (92%), single group (65%), nonrandomized (51%) and almost half were GBM trials. Only 10% involved a biologic and only 2.2% involved a double-blind randomized trial design. RESULTS: With this comparative literature review we conclude that our autologous cell product is uniquely antigen-inclusive and antigen-agnostic with a promising safety profile as well as unexpected clinical efficacy in our published phase 1b trial. We have since designed a rigorous double-blinded add-on placebo-controlled trial involving our implantable biologic drug device. We conclude that IGV-001 provides a novel immunotherapy platform for historically intransigent ndGBM in this ongoing phase 2b trial (NCT04485949).

A biologic-device combination product delivering tumor-derived antigens elicits immunogenic cell death-associated immune responses against glioblastoma.

BACKGROUND: IGV-001 is a personalized, autologous cancer cell-based immunotherapy conceived to deliver a tumor-derived antigenic payload in the context of immunostimulatory signals to patients with glioblastoma (GBM). IGV-001 consists of patient-derived GBM cells treated with an antisense oligodeoxynucleotide against insulin-like growth factor 1 receptor (IGF1R) and placed in proprietary biodiffusion chambers (BDCs). The BDCs are then exposed to 5-6 Gy radiation and implanted at abdominal sites for ~48 hours. IGV-001 has previously been shown to be generally safe with promising clinical activity in newly diagnosed GBM patients. METHODS: Mouse (m) or human (h) variants of IGV-001 were prepared using GL261 mouse GBM cells or human GBM cells, respectively. BDCs containing vehicle or mIGV-001 were implanted in the flanks of C57BL/6 albino female mice in preventative and therapeutic experiments, optionally in combination with a programmed cell death 1 (PD-1) blocker. Bioactivity of the general approach was also measured against hepatocellular carcinoma Hepa 1-6 cells. Mice were followed for the growth of subsequently implanted or pre-existing tumors and survival. Draining lymph nodes from mice receiving mIGV-001 were immunophenotyped. mIGV-001 and hIGV-001 were analyzed for extracellular ATP and high mobility group box 1 (HMGB1) as indicators of immunogenic cell death (ICD), along with flow cytometric analysis of viability, surface calreticulin, and reactive oxygen species. Stress and cell death-related pathways were analyzed by immunoblotting. RESULTS: IGV-001 causes oxidative and endoplasmic reticulum stress in GL261 cells, resulting in a cytotoxic response that enables the release of antigenic material and immunostimulatory, ICD-associated molecules including ATP and HMGB1 from BDCs. Immunophenotyping confirmed that IGV-001 increases the percentage of dendritic cells, as well as effector, and effector memory T cells in BDC-draining lymph nodes. Consistent with these observations, preventative IGV-001 limited tumor progression and extended overall survival in mice intracranially challenged with GL261 cells, a benefit that was associated with an increase in tumor-specific T cells with effector features. Similar findings were obtained in the Hepa 1-6 model. Moreover, therapeutically administered IGV-001 combined with PD-1 delayed progression in GBM-bearing mice. CONCLUSIONS: These results support treatment with IGV-001 to induce clinically relevant ICD-driven anticancer immune responses in patients with GBM.

Natural killer cells activity against multiple myeloma cells is modulated by osteoblast-induced IL-6 and IL-10 production.

Background: Natural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected cells and tumor cells without prior sensitization. The human NK-92MI cell line is among the most widely used NK cell in preclinical research studies and has also been approved for clinical applications. Previous studies have shown that osteoblasts (OSB) confer drug resistance in multiple myeloma (MM) and other cancers that metastasize to the bone marrow. Aim: We evaluated here how OSB, which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92MI cells against the MM.1S multiple myeloma cell line. Methods: The osteoblastic niche was recapitulated with either the osteoblastic cell line hFOB 1.19 (hFOB) or primary osteoblasts (P-OSB) derived from surgical resections. Time-lapse imaging was utilized to quantify changes in MM.1S cell viability under different conditions, including: (1) Co-culture of MM.1S with NK92MI cells, (2) triple-culture of hFOB or P-OSB with MM.1S and NK-92MI, and (3) MM.1S or NK-92MI cells primed with OSB-derived supernatant. Cytokine analysis was conducted to quantify potential secreted factors associated with the protective effects of OSB. Results: The physical presence of OSB hindered the activity of NK-92MI cells, resulting in the increased viability of MM.1S compared to co-cultures which lacked OSB. This observation was accompanied by reduced perforin and granzyme A secretion from NK-92MI cells. Contact of OSB and NK-92MI cells also induced interleukin 6 (IL-6) and interleukin 10 (IL-10) production; two cytokines which are known to impair the NK cell immunity against MM and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. Conclusions: We demonstrated here that OSB can negatively impact the activity of NK cells against MM. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell-mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the effectiveness of this potent immunotherapeutic approach.

Challenges and Opportunities for Immunotherapeutic Intervention against Myeloid Immunosuppression in Glioblastoma.

Glioblastoma multiforme (GBM), the most common and deadly brain cancer, exemplifies the paradigm that cancers grow with help from an immunosuppressive tumor microenvironment (TME). In general, TME includes a large contribution from various myeloid lineage-derived cell types, including (in the brain) altered pathogenic microglia as well as monocyte-macrophages (Macs), myeloid-derived suppressor cells (MDSC) and dendritic cell (DC) populations. Each can have protective roles, but has, by definition, been coopted by the tumor in patients with progressive disease. However, evidence demonstrates that myeloid immunosuppressive activities can be reversed in different ways, leading to enthusiasm for this therapeutic approach, both alone and in combination with potentially synergistic immunotherapeutic and other strategies. Here, we review the current understanding of myeloid cell immunosuppression of anti-tumor responses as well as potential targets, challenges, and developing means to reverse immunosuppression with various therapeutics and their status. Targets include myeloid cell colony stimulating factors (CSFs), insulin-like growth factor 1 (IGF1), several cytokines and chemokines, as well as CD40 activation and COX2 inhibition. Approaches in clinical development include antibodies, antisense RNA-based drugs, cell-based combinations, polarizing cytokines, and utilizing Macs as a platform for Chimeric Antigen Receptors (CAR)-based tumor targeting, like with CAR-T cells. To date, promising clinical results have been reported with several of these approaches.

Microfluidic device engineered to study the trafficking of multiple myeloma cancer cells through the sinusoidal niche of bone marrow.

Multiple myeloma (MM) is an incurable B cell malignancy characterized by the accumulation of monoclonal abnormal plasma cells in the bone marrow (BM). It has been a significant challenge to study the spatiotemporal interactions of MM cancer cells with the embedded microenvironments of BM. Here we report a microfluidic device which was designed to mimic several physiological features of the BM niche: (1) sinusoidal circulation, (2) sinusoidal endothelium, and (3) stroma. The endothelial and stromal compartments were constructed and used to demonstrate the device's utility by spatiotemporally characterizing the CXCL12-mediated egression of MM cells from the BM stroma and its effects on the barrier function of endothelial cells (ECs). We found that the egression of MM cells resulted in less organized and loosely connected ECs, the widening of EC junction pores, and increased permeability through ECs, but without significantly affecting the number density of viable ECs. The results suggest that the device can be used to study the physical and secreted factors determining the trafficking of cancer cells through BM. The sinusoidal flow feature of the device provides an integral element for further creating systemic models of cancers that reside or metastasize to the BM niche.

Pumpless microfluidic device with open top cell culture under oscillatory shear stress.

Here we developed a 96-well plate-based pumpless microfluidic device to mimic bidirectional oscillatory shear stress experienced by osteoblasts at the endosteal niche located at the interface between bone and bone marrow. The culture device was designed to be high-throughput with 32 open top culture chambers for convenient cell seeding and staining. Mathematical modeling was used to simulate the control of oscillatory shear stress with the peak stress in the range of 0.3 to 50 mPa. Osteoblasts, cultured under oscillatory shear stress, were found to be highly viable and significantly aligned along the direction of flow. The modeling and experimental results demonstrate for the first time that cells can be cultured under controllable oscillatory shear stress in the open top culture chamber and pumpless configurations.

Bifunctional Au-templated RNA nanoparticles enable direct cell uptake detection and GRP75 knockdown in prostate cancer.

Nucleic acids templated on gold (Au) surfaces have led to a wide range of functional materials ranging from microarrays, sensors and probes in addition to drug delivery and treatment. In this application, we describe a simple and novel method for templating amino-functionalized RNA onto Au surfaces and their self-assembly into small, discrete nanoparticles. In our method, sample hybridization with a complementary RNA strand with and without a fatty acid (palmitamide) appendage produced functionalized double-stranded RNA on the Au surface. The resulting Au-functionalized RNA particles were found to be stable under reducing conditions according to UV-Vis spectroscopy. Sample characterization by DLS and TEM confirmed self-assembly into primarily small (∼10-40 nm) spherical shaped nanoparticles expected to be amenable to cell biology. However, fluorescence emission (λexc: 350 nm, λem: 650 nm) revealed radiative properties which limited cell uptake detection. Introduction of FITC within the Au-functionalized RNA particles produced a bifunctional probe, in which FITC fluorescence emission (λexc: 494 nm, λem: 522 nm) facilitated cell uptake detection, in a time-dependent manner. The dual encapsulation-release profiles of the FITC-labeled Au-functionalized RNA particles were validated by time-dependent UV-Vis spectroscopy and spectrofluorimetry. These experiments respectively indicated an increase in FITC absorption (λabs: 494 nm) and fluorescence emission (λem: 522 nm) with increased sample incubation times, under physiological conditions. The release of Au-functionalized siRNA particles in prostate cancer (PC-3) cells resulted in concomitant knockdown of GRP75, which led to detectable levels of cell death in the absence of a transfection vector. Thus, the formulation of stable, small and discrete Au-functionalized RNA nanoparticles may prove to be valuable bifunctional probes in the theranostic study of cancer cells.

Size Matters: Arginine-Derived Peptides Targeting the PSMA Receptor Can Efficiently Complex but Not Transfect siRNA.

Oligoarginine sequences conjugated to a short cancer-targeting peptide (CTP) selective for the prostate-specific membrane antigen (PSMA) receptor was developed for selective small interfering RNA (siRNA) delivery to a human metastatic/castration-resistant prostate cancer (PCa) cell line, which expresses PSMA on the surface. The PSMA-Rn (n = 6 and 9) peptides were synthesized by solid-phase peptide synthesis, characterized by liquid chromatography-mass spectrometry (LC-MS) and condensed with glucose-regulated protein (GRP)-silencing siRNAs. Native gels showed formation of stable CTP:siRNA ionic complexes. Furthermore, siRNA release was effected by heparin competition, supporting the peptides' capabilities to act as condensing and releasing agents. However, dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies revealed large anionic complexes that were prone to aggregation and limited cell uptake for RNAi activity. Taken together, these data support the notion that the development of efficient peptide-based siRNA delivery systems is in part contingent on the formulation of discrete nanoparticles that can effectively condense and release siRNA in cells.

Pumpless platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation.

We report here a novel pumpless, 96-well plate-based platform for high-throughput dynamic multicellular culture and chemosensitivity evaluation. A gravity-driven flow strategy was developed to generate and sustain the flow rate of culture medium within 10% in the platform's 20 culture chambers. The ability of the platform to generate and sustain the medium flow was demonstrated by computational simulation, flow visualization, and ascertaining the previously known effect of flow-induced shear stress on the stimulated osteogenic differentiation of osteoblasts. The high-throughput utility of the platform was demonstrated by in situ cell staining and high content screening of chemosensitivity assays of multiple myeloma and osteoblast co-cultures. Endpoint characterization and data analyses for all 20 culture chambers required less than 1 hour.

Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths among American men. Unfortunately, there is no cure once the tumor is established within the bone niche. Although osteocytes are master regulators of bone homeostasis and remodeling, their role in supporting PCa metastases remains poorly defined. This is largely due to a lack of suitable ex vivo models capable of recapitulating the physiological behavior of primary osteocytes. To address this need, we integrated an engineered bone tissue model formed by 3D-networked primary human osteocytes, with conditionally reprogrammed (CR) primary human PCa cells. CR PCa cells induced a significant increase in the expression of fibroblast growth factor 23 (FGF23) by osteocytes. The expression of the Wnt inhibitors sclerostin and dickkopf-1 (Dkk-1), exhibited contrasting trends, where sclerostin decreased while Dkk-1 increased. Furthermore, alkaline phosphatase (ALP) was induced with a concomitant increase in mineralization, consistent with the predominantly osteoblastic PCa-bone metastasis niche seen in patients. Lastly, we confirmed that traditional 2D culture failed to reproduce these key responses, making the use of our ex vivo engineered human 3D bone tissue an ideal platform for modeling PCa-bone interactions.

GRP78 modulates cell adhesion markers in prostate Cancer and multiple myeloma cell lines.

BACKGROUND: Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment. METHODS: N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells. RESULTS: GRP78 KD led to concomitant downregulation of N-cad in both tumors types. In PC3 cells, GRP78 KD significantly decreased E-cadherin (E-cad) expression likely associated with the induction in TGF-ß1 expression. Furthermore, GRP78 KD also triggered drastic changes in PC3 cells morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cad expression. CONCLUSION: This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

Enhanced Cancer Theranostics with Self-Assembled, Multilabeled siRNAs.

The integration of therapy and diagnostics, termed "theranostics", has recently gained widespread utility in the development of new and improved therapeutics that effectively diagnose and treat diseases, such as cancer. In this study, the covalent attachment of multiple fluorescent labels (i.e., fluorescein isothiocyanate (FITC)) to a wide range of siRNAs, including those adopting linear, V- and Y-shape nanostructures, was successfully accomplished by solid-phase bioconjugation for monitoring cell uptake, co-localization, and biological activity in cell culture. The FITC-labeled higher-order V- and Y-shape siRNAs maintained the requisite hybrid stabilities and A-type helical structures for invoking RNAi activity. The FITC-siRNA hybrids with sense-strand modifiers enabled efficient mRNA knockdown (∼50-90%), which also translated to increased cell death (∼20-95%) in a bone metastatic prostate cancer cell line, over a 72 h incubation period. Significantly, the Y-shaped siRNA containing three FITC probes enhanced fluorescent signaling relative to the siRNA constructs containing single and double fluorophores while retaining potent knockdown and cell death effects post-transfection. Taken together, this data highlights the theranostic utility of the multilabeled FITC-siRNA constructs for potential cancer gene therapy applications.

Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose-Regulated Chaperones in Prostate Cancer Cells.

The emerging field of RNAi nanotechnology has led to rapid advances in the applications of siRNAs in chemical biology, medicinal chemistry, and biotechnology. In our RNAi approach, bioconjugation of linear, V-, and Y-shaped RNA templates were designed using a series of saturated and unsaturated fatty acids to improve cell uptake and knockdown efficacy of the oncogenic glucose regulated proteins (GRPs) in prostate (PC-3) cancer cells. An optimized HCTU-coupling procedure was developed for tagging variable saturated and unsaturated fatty acids onto the 5'-ends of linear and V-shaped RNA templates that were constructed by semiautomated solid phase RNA synthesis. Hybridization and self-assembly of complementary strands yielded linear, V-, and Y-shaped fatty acid-conjugated siRNAs which were characterized by native PAGE. CD spectroscopy confirmed their A-type helix conformations. RP IP HPLC provided trends in amphiphilic properties, whereas DLS and TEM confirmed multicomponent self-assembled structures that were prone to aggregation. Subsequently, the fatty acid conjugated siRNA bioconjugates were tested for their RNAi activity by direct transfection within PC-3 cells known to overexpress oncogenic GRP activity. The siRNA bioconjugates with sense strand modifiers provided more potent GRP knockdown relative to the antisense modified siRNAs, but to a lesser extent when compared to the unconjugated siRNA controls that were transfected with the commercial Trans-IT X2 dynamic delivery system. Flow cytometry revealed that the latter may be at least in part attributed to limited cell uptake of the fatty acid conjugated siRNAs. Nonetheless, these new constructs represent an entry point in modifying higher-order siRNA constructs that may lead to the generation of more efficient siRNA bioconjugates for screening important oncogene targets and for cancer gene therapy applications.

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction.

Osteocytes, residing as 3-dimensionally (3D) networked cells in bone, are well known to regulate bone and mineral homeostasis and have been recently implicated to interact with cancer cells to influence the progression of bone metastases. In this study, a bone tissue consisting of 3D-networked primary human osteocytes and MLO-A5 cells was constructed using: (1) the biomimetic close-packed assembly of 20-25µm microbeads with primary cells isolated from human bone samples and MLO-A5 cells and (2) subsequent perfusion culture in a microfluidic device. With this 3D tissue construction approach, we replicated ex vivo, for the first time, the mechanotransduction function of human primary osteocytes and MLO-A5 cells by correlating the effects of cyclic compression on down-regulated SOST and DKK1 expressions. Also, as an example of using our ex vivo model to evaluate therapeutic agents, we confirmed previously reported findings that parathyroid hormone (PTH) decreases SOST and increases the ratio of RANKL and OPG. In comparison to other in vitro models, our ex vivo model: (1) replicates the cell density, phenotype, and functions of primary human osteocytes and MLO-A5 cells and (2) thus provides a clinically relevant means of studying bone diseases and metastases.

Hypoxic Three-Dimensional Cellular Network Construction Replicates Ex Vivo the Phenotype of Primary Human Osteocytes.

Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.

Ex vivo construction of human primary 3D-networked osteocytes.

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20-25µm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D-constructed cells to form cellular network via processes with an average cell-to-cell distance of 20-25µm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D-networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

Tissue Engineering Platforms to Replicate the Tumor Microenvironment of Multiple Myeloma.

We described here the manufacturing and implementation of two prototype perfusion culture devices designed primarily for the cultivation of difficult-to-preserve primary patient-derived multiple myeloma cells (MMC). The first device consists of an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment. The second platform is a 96-well plate-modified perfusion culture device that can be utilized to reconstruct several tissue and tumor microenvironments utilizing both primary human and murine cells. This culture device was designed and fabricated specifically to: (1) enable the preservation of primary MMC for downstream use in biological studies and chemosensitivity analyses and, (2) provide a high-throughput format that is compatible with plate readers specifically seeing that this system is built on an industry standard 96-well tissue culture plate.

Ex vivo 3D osteocyte network construction with primary murine bone cells.

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Microbeads-Guided Reconstruction of 3D Osteocyte Network during Microfluidic Perfusion Culture.

Osteocytes reside as 3-dimensionally networked cells in the lacunocanalicular structure of bones, and function as the master regulators of homeostatic bone remodeling. We report here, for the first time to our best knowledge, the use of a biomimetic approach to reconstruct the 3D osteocyte network with physiological relevant microscale dimensions. In this approach, biphasic calcium phosphate microbeads were assembled with murine early osteocytes (MLO-A5) to provide an initial mechanical framework for 3D network formation and maintenance during long-term perfusion culture in a microfluidic chamber. The microbead size of 20-25 µm was used to: (1) facilitate a single cell to be placed within the interstitial space between the microbeads, (2) mitigate the proliferation of the entrapped cell due to its physical confinement in the interstitial site, and (3) control cell-to-cell distance to be 20-25 µm as observed in murine bones. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads within 3 days of culture. The entrapped cells produced significant mineralized extracellular matrix to fill up the interstitial spaces, resulting in the formation of a dense tissue structure during the course of 3-week culture. We found that the time-dependent osteocytic transitions of the cells exhibited trends consistent with in vivo observations, particularly with high expression of Sost gene, which is a key osteocyte-specific marker for the mechanotransduction function of osteocytes. In contrast, cells cultured in 2D well-plates did not replicate in vivo trends. These results provide an important new insight in building physiologically relevant in vitro bone tissue models.