Spherical Multicellular Aggregates Research Articles

Embedded bioprinted multicellular spheroids modeling pancreatic cancer bioarchitecture towards advanced drug therapy.

The desmoplastic bioarchitecture and microenvironment caused by fibroblasts have been confirmed to be closely related to the drug response behavior of pancreatic ductal adenocarcinoma (PDAC). Despite the extensive progress in developing PDAC models as in vitro drug screening platforms, developing efficient and controllable approaches for the construction of physiologically relevant models remains challenging. In the current study, multicellular spheroid models that emulate pancreatic cancer bioarchitecture and the desmoplastic microenvironment are bioengineered. An extrusion-based embedded dot bioprinting strategy was established to fabricate PDAC spheroids in a one-step process. Cell-laden hydrogel beads were directly deposited into a methacrylated gelatin (GelMA) suspension bath to generate spherical multicellular aggregates (SMAs), which further progressed into dense spheroids through in situ self assembly. By modulating the printing parameters, SMAs, even from multiple cell components, could be manipulated with tunable size and flexible location, achieving tunable spheroid patterns within the hydrogel bath with reproducible morphological features. To demonstrate the feasibility of this printing strategy, we fabricated desmoplastic PDAC spheroids by printing SMAs consisting of tumor cells and fibroblasts within the GelMA matrix bath. The produced hybrid spheroids were further exposed to different concentrations of the drug gemcitabine to verify their potential for use in cell therapy. Beyond providing a robust and facile bioprinting system that enables desmoplastic PDAC bioarchitecture bioengineering, this work introduces an approach for the scalable, flexible and rapid fabrication of cell spheroids or multi-cell-type spheroid patterns as platforms for advanced drug therapy or disease mechanism exploration.

Development, characterization, and applications of multi-material stereolithography bioprinting

As a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure–function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-β1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments.

Evaluation of a Hybrid Artificial Liver Module Based on a Spheroid Culture System of Embryonic Stem Cell-Derived Hepatic Cells

Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.

Morphological and functional behaviors of rat hepatocytes cultured on single-walled carbon nanotubes

This study describes the morphological and functional behaviors of rat hepatocytes on single-walled carbon nanotube (CNT)-coated surfaces. Although the hydrophobic characteristics of CNT-coated surfaces increased with increasing CNT density, hepatocyte adhesion decreased, indicating that the interaction between hepatocytes and CNTs is weak. We found that hepatocytes on a CNT-coated surface gradually gather together and form spheroids (spherical multicellular aggregates). These spheroids exhibit compact spherical morphology with a smooth surface and express connexin-32, an intracellular communication molecule. In contrast, collagen treatment in conjunction with the CNT-coated surface improved hepatocyte adhesion, and the cells maintained a monolayer configuration throughout the culture period. The albumin secretion and ammonia removal activities of hepatocyte spheroids were maintained at elevated levels for at least 15 days of culturing as compared with hepatocyte monolayers. These results indicate that CNTs can be used for the formation and long-term culture of hepatocyte spheroids.

Comparative Analysis of Gene Expression in Rat Liver Tissue and Monolayer- and Spheroid-Cultured Hepatocytes

A culture system with spherical multicellular aggregates (spheroids), which are formed by the rearrangement and compaction of cell aggregates, is reported to be more useful than the traditional monolayer culture system for the culture of primary hepatocytes. By performing real-time polymerase chain reaction, we analyzed the expression of genes encoding key molecules involved in liver-specific functions, namely, cell adhesion molecules (integrin 3, cadherin 1 and connexin 32), transcription factors (hepatic nuclear factor 4α and CCAAT/enhancer-binding protein β), protein and metabolic enzymes (albumin, glucose-6-phosphatase, tryptophan 2,3-dioxygenase, arginase 1 and cytochrome P450 7A1) and transporters (organic anion transporting peptide 1, multidrug resistance-associated protein 2 and bile salt export pump), in spheroids derived from rat hepatocytes. Further, we compared these expression levels with those in a hepatocyte monolayer and in liver tissue. Only the gene encoding glucose-6-phosphatase (required for sugar metabolism) was expressed at a similar level in both the monolayer culture and liver tissue for 10 days of culture; the expression of all the other genes in the monolayer culture either rapidly decreased or completely disappeared as the culture duration increased. Although the expression levels of all the genes in the spheroids tended to decrease gradually with culture time, they were consistently higher than those in the monolayer culture for at least 10 days of culture. These results suggest that hepatocyte spheroids acquire intercellular organization and largely maintain many intercellular metabolic functions. Thus, the hepatocyte spheroid culture system seems to be promising for various in vitro cell-based assays.

Hepatic differentiation of mouse embryonic stem cells in a three-dimensional culture system using polyurethane foam

Embryonic stem (ES) cells are a type of pluripotent stem cell line isolated from the inner cell mass of blastocysts and characterized by an almost unlimited self-renewal capacity and differentiation potential in vitro into multiple cell lineages. Therefore the use of ES cells has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional matured cells from ES cells in large quantities. In this study, we applied polyurethane foam (PUF)/spheroid culture, which enables spontaneous spheroid formation and mass cultivation of cultured cells, to mouse ES cells for hepatic differentiation. Mouse ES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF within 1 d. To induce hepatic differentiation, specific growth factors were added to the culture medium. Mouse ES cells proliferated by day 20, and high cell density (about 1.0 x 10(8) cells/cm(3)-PUF) was achieved. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, albumin and tryptophan 2,3-dioxygenase. The activity of ammonia removal of mouse ES cells per unit volume of the module was detected by day 21 and increased with culture time. Maximum expression levels were comparable to those of primary mouse hepatocytes. Mouse ES cells could express liver-specific functions at high level because of the high cell density culture and hepatic differentiation. These results suggest that the PUF/spheroid culture method could be useful to develop mass differentiation cultures.

Hepatic Differentiation of Mouse Embryonic Stem Cells in a Bioreactor Using Polyurethane/Spheroid Culture

Background We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. Methods The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. Results mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 × 10 8 cells/cm 3 PUF). Differentiating ES cells expressed endodermal-specific genes such as α-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. Summary mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

ラクトース修飾絹フィブロイン基材上における初代培養ラット肝細胞のスフェロイド形成と維持

We examined the formation and maintenance of spherical multicellular aggregates (spheroids) of rat primary hepatocytes on dishes coated with lactose-silk fibroin conjugates bearing galactose residues (Lac-CY-SF) as substrate materials. Rat hepatocytes that had attached onto the conjugate-coated dishes were subsequently cultured in a medium supplemented with epidermal growth factor (EGF) and insulin. After the rat hepatocytes extended flat on the conjugate-coated dishes in 2 days of culture, these hepatocytes formed spheroids about 100 to 300 μm in diameter at day 4. After 6 days of culture the spheroids were maintained without any obvious change in size on the conjugate-coated dishes. However, the detachment of the spheroids from the conjugate-coated dishes began from day 8, and the spheroids clearly shrank at day 10. These results suggested that hepatocyte spheroid formation was induced on the Lac-CY-SF conjugate-coated dishes in the presence of EGF and insulin, but the conjugate-coated dishes were not capable of keeping the spheroids for more than 5 days.

Hepatocyte spheroid culture on a polydimethylsiloxane chip having microcavities

A two-dimensional microarray technique of spherical multicellular aggregates (spheroids) using a microfabricated polydimethylsiloxane (PDMS) chip and the expression of liver-specific functions of primary rat hepatocytes on the chip were investigated. The PDMS chip, which was fabricated by a photolithography-based technique, consisted of approximately 2500 cylindrical microcavities (approximately 1100 cavities/cm2) in a triangular arrangement of 330 μm pitch on a PDMS plate (20 × 20 mm); each cavity measured 300 μm in diameter and 100 μm in depth. Most hepatocytes on the PDMS chip gradually gathered and subsequently formed a single spheroid in each cavity until 3 days of culture. A part of the spheroid was attached to the bottom or wall surface of the microcavity, and the spheroid configuration was maintained for at least 14 days of culture. Albumin secretion, ammonia removal and ethoxyresorufin O-dealkylase (EROD) activity, which is a cytochrome P-450-dependent reaction, of hepatocytes on the PDMS chip were higher than those of a monolayer dish or a flat PDMS dish without microcavities, and were maintained for at least 10 days of culture. The spheroid microarray technique appears to be promising in the development of cell chips and microbioreactors.

Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.

This article describes a novel method for preparing several spherical multicellular aggregates (spheroids) that have almost the same diameter on a microfabricated chip. The chip, fabricated by a simple method that uses a micromilling system, consisted of several thousand cavities, 100-500 microm in diameter, in a triangular arrangement on a polystyrene plate. Although no spheroid was formed on any chip when cultured under stationary conditions, hepatocytes formed spheroids in the cavities when turning force was applied with a rotary shaker. Especially on the chip with cavities 300 microm in diameter, one spheroid formed in each cavity; an orderly array of spheroids (1100 spheroids/cm(2)) of almost the same diameter was constructed. Ammonia removal and albumin secretion by the spheroids on the chip continued to occur at initial levels for at least 14 days of culture. Enzyme P-450 activity of the spheroids was also maintained and detected on this transparent chip by fluorescence of resorufin converted from ethoxyresorufin. The spheroid microarray chip seems to be a promising cellular platform for various biomedical applications such as in cell-based biosensors for toxicological and pharmacological examinations, and in bioartificial livers.

Isolated Hepatocytes versus Hepatocyte Spheroids: In Vitro Culture of Rat Hepatocytes

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 +/- 4.4%), while in G2 it was 72.1 +/- 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.

Mass Preparation of Primary Porcine Hepatocytes and the Design of a Hybrid Artificial Liver Module using Spheroid Culture for a Clinical Trial

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63+/-78 x 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) total cells with a viability of 87.9+/-4.4% (mean +/- SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387+/-89 x 10(8) hepatocytes with a viability of 87.1+/-6.9% and a purity of 93.6+/-2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 10(8) porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.

Formation of porcine hepatocyte spherical multicellular aggregates (spheroids) and analysis of drug metabolic functions.

Porcine hepatocytes are used in the hybrid artificial liver support system that we are developing because of their high level of liver functions in vitro and because human hepatocytes can not be used in Japan for ethical reasons. Spherical multicellular aggregates or spheroids have been found to be effective in vitro for long-term maintenance of liver functions. Therefore, we formed spherical multicellular aggregates (spheroids) of primary porcine hepatocytes using a polyurethane foam (PUF) as a culture substratum and analyzed their drug metabolic functions in vitro. Primary porcine hepatocytes inoculated into the pores of a flat PUF plate (25 x 25 x 1 mm), spontaneously formed spheroids within the range of 100 to 150 mum in diameter 24 to 36 h after inoculation. The formed spheroids were attached to the bottom surface of the PUF pores, and their morphology and viability were maintained for more than 12 days. The P-450 activity in the spheroids of porcine hepatocytes was demonstrated by detecting production of monoethylglycinexylidide from lidocaine. In addition, the conjugation enzyme activity was demonstrated by detecting glucuronidation and sulfation of acetaminophen. These activities were maintained for 12 days at a level twice as high as in the monolayer culture. This result shows that the porcine hepatocyte spheroids formed by using PUF can maintain the drug metabolic functions important in a hybrid artificial liver device. Consequently, culturing porcine hepatocyte spheroids using PUF seems to be promising for development of a hybrid artificial liver.

The formation of a spheroid of primary hepatocytes and the expression of liver-specific functions depend on the characteristics of polyurethane foam

Rapid formation of spherical multicellular aggregates (spheroids) of hepatocytes and expression of liver-specific functions are important in developing a hybrid artificial liver. The relation between these points and the surface characteristics of the culture substratum are investigated in this article. Polyurethane foam (PUF) was used as a culture substratum for hepatocytes. Rat, dog, and porcine primary hepatocytes spontaneously formed a spheroid within 1.5 days in the pores of a hydrophilic PUF with a contact angle of 53.4±2.7°. Rat and dog primary hepatocytes formed a spheroid within 3 and 7 days of culture, respectively, in hydrophobic PUF with a contact angle of about 100°. The rates of albumin secretion and ammonia metabolism at 5 days of culture of porcine hepatocytes were 31 μg of albumin/106 nuclei/day and 0.018 μmol of NH3/106 nuclei/h, respectively, about 2 times higher than the values with hydrophobic PUF. It is very important to control the hydrophilicity of the PUF surface to develop an effective artificial liver module.

Hepatocyte Spheroids in Polyurethane Foams: Functional Analysis and Application for a Hybrid Artificial Liver

Spherical multicellular aggregates (spheroids) of adult rat hepatocytes were spontaneously formed in the pores of polyurethane foam (PUF) as a culture substratum. Highly differentiated functions of...

Formation of a spherical multicellular aggregate (spheroid) of animal cells in the pores of polyurethane foam as a cell culture substratum and its application to a hybrid artificial liver

Monkey kidney cells (Vero), human embryonic kidney cells (293), human liver cells (PLC/PRF/5), and primary rat, dog, and porcine hepatocytes formed spherical multicellular aggregates (spheroids) in the pores of polyurethane foam which was used as a cell culture substratum. These spheroids of various cell types express high cell activity for a long period. A practical hybrid artificial liver support system composed of a multi-capillary polyurethane foam packed-bed type cell culture module including primary hepatocyte spheroids was developed. The success of the system is indicated by an 80% recovery rate in hepatic failure rats which died in control experiments.

Multinucleated giant cells in primary cultures derived from canine bone marrow--evidence for formation of putative osteoclasts.

Mononucleated cells derived from canine bone marrow were maintained in vitro for up to 6 weeks. The culture characteristics and development of these cells were evaluated by histological, ultrastructural and histochemical methods. Within 1 week the cells had fused together to form flattened, multinucleated cells. Further fusing with one another and other mononucleated cells produced large (diameters more than 300 microns), multinucleated cells which frequently contained more than 50 nuclei per cell and exhibited ultrastructural and histochemical features that were strikingly similar to those displayed by osteoclasts. The confluent monolayer of mono- and multinucleated cells present at 4 weeks had, by the sixth week, become altered such that fibroblast overgrowth obliterated all other cells. During the development of the culture adipocytes became differentiated from mononuclear cells and frequently were located within spherical multicellular aggregates (spheroids). Functional assessments were employed to investigate whether the multinucleated cells generated in this way, represented osteoclast-like cells, or alternatively, were related to macrophage polykarya as found in foreign body granulomata in vivo. Neither resorption pits on sperm whale dentine slivers (diagnostic of osteoclasts), nor formation of granulomata in vitro, were observed. We believe that the present results indicate that the multinucleated cells generated from canine bone marrow mononuclear precursors in vitro, merit designation as osteoclast-like cells. Definitive characterisation however, must await further functional assessments of hormone responsiveness.

High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam

Adult rat hepatocytes formed spherical multicellular aggregates (spheroids) when they were cultured in the pores of polyurethane foam (PUF). The diameter of the spheroids was within the range 100-200 microns. These spheroids partly attached and immobilized in the PUF pores for at least 2 weeks. The albumin production rate by the spheroids increased up to 17.0 micrograms/10(6) nuclei per day during the first 6 days and maintained at a high level for 2 weeks. In contrast, the albumin production rate by the monolayer markedly decreased after 3 days. The spheroid culture using PUF seems to be a convenient and simple method for maintaining some differentiated functions of hepatocytes and for making a bioreactor using the function of spheroids.