Spheroid Culture of Mammalian Olfactory Receptor Neurons: Potential Applications for a Bioelectronic Nose

The olfactory system can detect many odorants with high sensitivity and selectivity based on the expression of nearly a thousand types of olfactory receptors (ORs) in olfactory receptor neurons (ORNs). These ORs have a dynamic odorant detection range and contribute to signal encoding processes in the olfactory bulb (OB). To harness the capabilities of the olfactory system and develop a biomimetic sensor, stable culture and maintenance of ORNs are required. However, in vitro monolayer culture models have several key limitations: i) short available period of cultured neurons, ii) low cultural efficiency, and iii) long-term storage challenges. This study aims to develop a technique: i) to support the spheroid culture of primary ORN precursors facilitating stable maintenance and long-term storage, and ii) to demonstrate the viability of ORN spheroid culture in developing an olfactory system mimetic bioelectronic nose. Recombinant protein (REP; TGPG[VGRGD(VGVPG)₆]₂₀WPC) was used to form the ORN spheroids. Spheroid formation enabled preservation of primary cultured ORNs without a significant decrease in viability or the expression of stemness markers for ten days. Physiological characteristics of the ORNs were verified by monitoring intracellular calcium concentration upon odorant mixture stimulation; response upon odorant stimulation were observed at least for ten days in these cultivated ORNs differentiated from spheroids. Coupling ORNs with multi electrode array (MEA) enabled the detection and discrimination of odorants by analyzing the electrical signal patterns generated following odorant stimulation. Taken together, the ORN spheroid culture process is a promising technique for the development of a bioelectronic nose and high-throughput odorant screening device.

The viability and property of rabbit corneal endothelial cells by spheroid culture / 中华实验眼科杂志

Background The construction of tissue-engineered corneal endothelium and corneal endothelial cells (CECs) therapy need abundant seed cells,so how to culture a large amount of CECs with high viability and original cell properties is an urgent issue to be solved.Objective This study was to establish three-dimensional spheroid culture of CECs and explore the cellular biological characteristics.Methods Primary rabbit CECs were isolated with trypsin and subcultured.Low attachment and shaking culture was applied to form CECs spheres.Cultured cells were identified under the inverted microscope.The surface features of the cells were examined under the scanning electron microscope.The viability of the cells were assayed by acridine orange (AO) staining and CCK-8 kit.Then CECs spheres were incubated to 6-well plate for 1 week,and immunofluorescence staining was used to identify the expression of zonula occludens-1 (ZO-1) and Na+/K+-ATPase in the cells.The cell proliferation value of spheroid culture method was compared with that of regular culture method.Results CECs grew into aggregation after cultured with the hexagonal or polygonal shape and tight connection among the cells.The cells converged into single layer and slabstone-like arrangement 1 week later.Cells migrated out of the CECs sphere and formed an uneven spherical surface.The living cells showed green fluorescence for AO with the survival rate 90％.The absorbance (A450) of the cells was 1.524±0.013 and 1.265 ±0.021 in the spherical culture group and conventional culture group,respectively,showing a significant difference between them (t =-3.436,P=0.010).The positive cells of ZO-1 and Na+/K+-ATPase showed the green fluorescence for FITC on cell membrane and blue fluorescence for DAPI on cell nucleus.Conclusions Spherical culture method maintains a high viability and proliferation ability of the cells and remains phenotype of CECs,which is superior to conventional culture method.This culture method provides better seeding CECs for the establishment of tissue engineering cornea endothelial layer and CECs therapy.

Optimal Number of Hepatocytes per Microcarrier in Spheroid Culture using Cytodex 3 Microcarrier

PURPOSE: The mass cultivation of functional hepatocytes is a key factor of a bioartificial liver. Combining spheroid and microcarrier cultures has been applied for enhancing the cell viability and metabolic activities. Hence, the optimal number of hepatocytes per microcarrier was investigated. METHODS: Firstly, spheroid cultures were carried out with 1 g Cytodex 3 microcarrier plus 2 x 10(9), 4 x 10(8) and 8 x 10(7) viable hepatocytes per flask. The numbers of hepatocytes per microcarrier were approximately 666.7, 133.3 and 26.7, respectively. The control group consisted of a spheroid culture of 4 x 10(8) hepatocytes without any microcarrier. According to the primary experimental results, spheroid cultures with 1 x 10(8) of hepatocytes plus 1 g, 2 g and 3 g of the Cytodex 3 microcarrier were performed. The numbers of hepatocytes per microcarrier were approximately 33.3, 16.7 and 11.1, respectively. The control group consisted of a spheroid culture of 1 x 10(8) hepatocytes. The cell viabilities were assayed using a Cell Counting Kit-8; with the albumin production assayed using ELISA. RESULTS: According to the primary experiment, the group consisting of 26.7 hepatocytes per microcarrier showed the highest viability (P<0.01). However, there was no statistical difference in the albumin production between the groups (P=0.744). The second Experiment showed the groups consisting of 11.1 and 16.7 hepatocytes per microcarrier had higher viabilities than the other hepatocyte and control groups (P<0.01). The albumin production was similar for each group (P=0.187). CONCLUSION: With respect to their application to a bioartificial liver, about 130 hepatocytes per microcarrier was appeared to be good for the mass cultivation of a hepatocytes spheroid culture using the Cytodex 3 microcarrier.

Isolation and Culture of Pig Hepatocyte in Large Scale for the Application of Bioartificial Liver System / 대한간학회지

BACKGROUND/AIMS: Acute hepatic failure is a serious problem. Its mortality reaches up to 80%. Only liver transplantation has been accepted as a definite treatment for patients with hepatic failure but shortage of donor organs is the main obstacle of this approach. A possible solution to this problem is a bioartificial liver system, perfusion of patients blood to isolated hepatocyte. In this study, we performed the isolation and culture of pig hepatocyte in large scale for the application of bioartificial liver system. METHODS: Hepatocyte isolation was performed by two-step collagenase method via portal vein perfusion in 10kg female pigs. After that, we compared the functional differences of the spheroid culture to the monolayer culture of hepatocyte. The viability and the function of hepatocyte were assessed using trypan-blue exclusion test and the measurement of the rate of ureagenesis and ammonia removal. RESULTS: The average viability and yield of hepatocyte were 86.8 +/- 8.0 % and 7.8 +/- 5.4 X 10(9), respectively. The spheroid culture was superior to the monolayer culture in functional aspect of hepatocyte, and their differences, especially for ammonia removal, were more apparent in parallel with culture time. CONCLUSIONS: For hepatocyte isolation, we obtained sufficient viability and yield of hepatocyte for clinical usage of bioartificial liver system. The function of hepatocyte seems to be better in the spheroid culture than in the monolayer culture. Further studies are needed for application of bioartificial liver system in clinical setting.