Encapsulation of rat hepatocyte spheroids for the development of artificial liver

Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver.     

Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids

Summary Freshly harvested rat hepatocytes form spheroids on uncoated positively charged polystyrene surfaces. Time lapse microscopy revealed that cell movement and reorganization were involved in spheroid formation. Ultrastructural evaluation using scanning and transmission electron microscopy indicated polarized cellular morphology and extensive cell-cell communication within spheroids. Bile canalicular structures were observed to surround each individual hepatocyte, forming an intricate three-dimensional continuous network of channels that appeared to end as pores/holes on the surface of the spheroid. The maintenance of differentiated cellular morphology coincided with preservation of hepatocyte viability and enhanced levels of tissue specific functions in spheroids.     

A novel method to prepare size-regulated spheroids composed of human dermal fibroblasts

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 mug/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 mum in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 mum in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests. (c) 1994 John Wiley & Sons, Inc.     

Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel

The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture. Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide. Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment system may facilitate the development of a bioartificial liver support device.     

A novel method to prepare multicellular spheroids from varied cell types

A simple method for preparing multicellular spheroids from varied cell types has been successfully developed by using a stepwise gradient surface in cell attachability or detachability. The surface was composed of poly-N-isopropylacrylamide (PNIPAAm), a temperature responsive polymer, as a cell detaching component, and collagen as a cell attaching component. The surface functions as a culture substratum at 37 degrees C; then, when lowering the temperature of culture medium, the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature (LCST; about 30 degrees C), but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. The stepwise gradient surface which consisted of six different sectors was prepared by exposing a surface of the PNIPAAm-collagen mixture to ultraviolet (UV) irradiation six times using a photomask, sliding the hole position in the photomask, and changing the energy of UV irradiation. This was because crosslinking of collagen depended on the energy of UV irradiation, then, cell attachability to and detachability from the surface were tightly controlled by changing the energy.The stepwise gradient surface allowed us to easily determine optimal surface conditions to obtain good cell attachment and detachment as a self-supporting sheet from the surface to prepare multicellular spheroids. According to the evaluation of the attachability and detachability of 23 cell types, the optimal surface condition remarkably depended on each cell type. The detached cells under optimal surface conditions, including fibroblasts, osteoblastic cells, smooth muscle cells, and measangial cells, which were very difficult to form spherioids using conventional methods, were able to form multicellular spheroids. The results clearly demonstrate that the above-described method for preparing multicellular spheroids can be applied to varied cell types. (c) 1995 John Wiley & Sons, Inc.     

Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Effects of degree of cell-cell contact on liver specific functions of rat primary hepatocytes

Cell-cell interaction and the extracellular matrix (ECM) are believed to play essential roles duringin vitro culturing of primary hepatocytes in the control of differentiation and in the maintenance of tissue specific functions. The objective of this study was to examine the effects of degree of cell-cell contact (DCC) on liver specific function of rat primary hepatocytes. Hepatocyte aggregates with various degrees of cell-cell contact,i.e., dispersed cells, longish aggregate, rugged aggregate, and smooth spheroid were obtained at 1, 5–6, 15–20, and 36–48 hrs, respectively in suspension cultures grown in spinner flasks embedded in Caalginate bead and collagen gel in order. The smooth spheroids displayed a decrease in viability and functional activities. This may result from mass transfer limitation and shear damage caused by agitation during aggregation. The rugged aggregate showed a higher viability and albumin secretion rate than the dispersed cells or the other aggregates. This result indicates the possible enhancement of a bioartificial liver's (BAL) performance using primary hepatocytes and the reduction in time to prepare a BAL through optimization of the immobilization time.     

Developing scalable cultivation systems of hepatic spheroids for drug metabolism via genomic and functional analyses

Spheroids, a widely used three-dimensional (3D) culture model, are standard in hepatocyte culture as they preserve long-term hepatocyte functionality and enhance survivability. In this study, we investigated the effects of three operation modes in 3D culture — static, orbital shaking, and under vertical bidirectional flow using spheroid forming units (SFUs) on hepatic differentiation and drug metabolism to propose the best for mass production of functionally enhanced spheroids. Spheroids in SFUs exhibited increased hepatic gene expression, albumin secretion, and cytochrome P450 3A4 (CYP3A4) activity during the differentiation period (12 days). SFUs advantages include facilitated mass production and a relatively earlier peak of CYP3A4 activity. However, CYP3A4 activity was not well maintained under dimethyl sulfoxide (DMSO)-free conditions (13–18 days), dramatically reducing drug metabolism capability. Continued shear stimulation without differentiation stimuli in assay conditions markedly attenuated CYP3A4 activity, which was less severe in static conditions. In this condition, SFU spheroids exhibited dedifferentiation characteristics, such as increased proliferation and Notch signaling genes. We found that the dedifferentiation could be overcome by using the serum-free medium formulation. Therefore, we suggest that SFUs represent the best option for the mass production of functionally improved spheroids and so the serum-free conditions should be maintained during drug metabolism analysis.     

Enhanced liver-specific functions of endothelial cell-covered hepatocyte hetero-spheroids

The performance of an extracorporeal bioartificial liver (BAL) support system depends on the functional activities of the hepatocytes immobilized in the system. One of the most promising techniques in retaining liver-specific functions is co-culturing hepatocytes with other cell types, such as epithelial cells, endothelial cells and dermal fibroblasts. Primary rat hepatocytes were suspension co-cultured with rat prostate endothelial cell line (RPEn) for 20 h in a spinner vessel to form hetero-spheroids, which contain the two types of the cells, i.e., hepatocytes and endothelial cells in the same spheroid. For the subsequent culture, the hetero-spheroids were entrapped in a Ca-alginate gel bead. From the results of incorporation efficiency test, it was found that RPEn cells have a significantly higher attachment affinity to hepatocytes than human dermal fibroblast and rat liver epithelial cells. We clearly found out that RPEn cells located on the surface of the hepatocyte spheroids from immunostained paraffin sections of the hetero-spheroids. Identical with in vivo liver tissue, laminin was stained at the surface of the hetero-spheroids. Ultrastructures of liver tissue, such as bile canaliculus-like and Disse’s space-like structures, were also found at the surface of the hetero-spheroids. In vivo liver tissue, in which hepatocytes were covered with sinusoidal endothelial cells, was partly mimicked by the endothelial cell-covered hepatocyte spheroids. And the hetero-spheroids showed significantly higher and stable albumin secretion and ammonia removal activities than pure spheroids for 12 days of observations.

Therefore, the endothelial cell-covered hepatocyte hetero-spheroids may offer a useful study model of epithelial–mesenchymal interactions and information about liver tissue engineering research as well as a substitute of a cell source of a BAL system.     

Maintenance of hepatocyte functions in coculture with hepatic stellate cells

Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine.     

Phenotype of hepatocyte spheroids in synthetic thermo-reversible extracellular matrix

Aggregates of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver-specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide (98 mole % in feed) and acrylic acid (poly(NiPAAm-co-AAc)), a thermo-reversible copolymer gel matrix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture period, the spheroids maintained higher viability and produced albumin and urea at a relatively constant rate, while the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion. Hepatocytes cultured as spheroids present a potentially useful three-dimensional cell culture system for application in a bioartificial liver device.     

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.     

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.     

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 × 109 porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.     

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: II. nitric oxide synthesis and related changes

Liver cells isolated from intact tissue can reaggregate to form three-dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the 'challenge' induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell-cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.     

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 × 25 × 1 mm), spontaneously formed spheroids within the range of 100 to 150 μm 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. This revised version was published online in August 2006 with corrections to the Cover Date.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.     

Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7

Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.     

Development of a microchannel emulsification process for pancreatic beta cell encapsulation

In this study, we developed a high-throughput microchannel emulsification process to encapsulate pancreatic beta cells in monodisperse alginate beads. The process builds on a stirred emulsification and internal gelation method previously adapted to pancreatic cell encapsulation. Alginate bead production was achieved by flowing a 0.5–2.5% alginate solution with cells and CaCO₃ across a 1-mm thick polytetrafluoroethylene plate with 700 × 200 μm rectangular straight-through channels. Alginate beads ranging from 1.5–3 mm in diameter were obtained at production rates exceeding 140 mL/hr per microchannel. Compared to the stirred emulsification process, the microchannel emulsification beads had a narrower size distribution and demonstrated enhanced compressive burst strength. Both microchannel and stirred emulsification beads exhibited homogeneous profiles of 0.7% alginate concentration using an initial alginate solution concentration of 1.5%. Encapsulated beta cell viability of 89 ± 2% based on live/dead staining was achieved by minimizing the bead residence time in the acidified organic phase fluid. Microchannel emulsification is a promising method for clinical-scale pancreatic beta cell encapsulation as well as other applications in the pharmaceutical, food, and cosmetic industries.     

An in situ method for cultivating microorganisms using a double encapsulation technique

The lack of cultured microorganisms represents a bottleneck for advancement in microbiology. The development of novel culturing techniques is, therefore, a crucial step in our understanding of microbial diversity in general, and the role of such diversity in the environment, in particular. This study presents an innovative method for cultivating microorganisms by encapsulating them within agar spheres, which are then encased in a polysulfonic polymeric membrane and incubated in a simulated or natural environment. This method stimulates growth of the entrapped microorganisms by allowing them access to essential nutrients and cues from the environment. It allows for the discovery of microorganisms from dilutions that are 10–100-fold greater than possible with conventional plating techniques. Analysis of microorganisms grown in such spheres incubated in and on a number of different substrates yielded numerous novel ribotypes. For example, spheres incubated on the mucus surface of a Fungiid coral yielded numerous ribotypes, with only 50% sharing similarity (85–96%) to previously identified microorganisms. This suggests that many of the species represent novel ribotypes. Hence, the technique reported here advances our ability to retrieve and successfully culture microorganisms and provides an innovative tool to access unknown microbial diversity.