Polyvinyl Formal Resin Research Articles

Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation.

Three-dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer-cultured cells and 3D cultures of established cancer cell lines (sphere culture) or patient-derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time-efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness-related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross-linked PVA can function in serum-free, long-term 3D cultures to support maintenance of sphere- or tumor-like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.

Stopping of ∼0.2–3.4MeV/amu 1H+ and 4He+ ions in polyvinyl formal

Abstract The stopping powers of polyvinyl formal resin for protons and alpha particles have been measured over the energy range ∼(0.2–3.4) MeV/amu with an overall relative uncertainty of less than 2.5% using the ion beam transmission method. The obtained results are discussed in comparison to scarce experimental data from the literature and to values calculated by the SRIM-2008 computer code assuming Bragg-Kleeman’s additivity rule of stopping powers. Departures from additivity attaining ∼5.5% and ∼3.0% for the proton and alpha particle data, respectively, are observed at moderate projectile velocities, increasingly as one evolves towards the stopping power maximum dominated by charge exchange effects. They are presumed to be mainly due to valence structure effects involving the C–H bonds of the studied polymer compound. The less pronounced deviation for alpha particles is probably due to the projectile screening effect that occurs in distant collisions tending to reduce the contribution of valence electrons in the low projectile velocity regime.

Immobilization of Escherichia coli cells using polyethyleneimine-coated porous support particles for l-aspartic acid production

Escherichia coli cells were efficiently immobilized in reticulated polyvinyl formal resin biomass support particles (BSPs) that had been simply autoclaved with a solution of a cationic polymer such as polyethyleneimine. When the immobilized E. coli cells containing aspartase were used as whole cell biocatalyst for l-aspartic acid production, they showed specific aspartase activity comparable to that of non-immobilized cells.

Immobilization of Escherichia coli cells using porous support particles coated with cationic polymers

The technique of cell immobilization using porous biomass support particles (BSPs), which is attractive from the point of view of simplicity and convenience, relies on the inherent ability of adhesive cells, as a consequence of their growth, to form films around the support material or the ability of flocculent cells to create flocs within the porous structure. In the present study, the immobilization of Escherichia coli cells using BSPs was investigated in shake-flask culture. The density of the cells immobilized within the BSPs was evaluated by measuring their intracellular lactate dehydrogenase (LDH) activity. Since E. coli K12 cells were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs with matrices of relatively small pores (pore diameter 60 microm), coating the surface of the BSPs with various polymers was examined as a way of promoting cell attachment. When positively charged polyamino acids such as poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly-L-ornithine were adsorbed onto the particle surface, they were found to increase the immobilized cell density, while neutral and negatively charged polyamino acids including poly-L-asparagine and poly-L-glutamic acid were not effective. These results indicate that E. coli cells can be efficiently immobilized in PVF resin BSPs by electrostatic interaction between the negatively charged ions of the cell surface and the positively charged polymers adsorbed onto the BSP surface. A significantly high immobilized cell density was also achieved by coating the surface of the BSPs with the synthetic polymeric amine polyethyleneimine.

Efficient production of recombinant protein in immobilized insect cell culture using serum-free basal media after baculovirus infection

To develop an efficient biological production process using the baculovirus–insect cell system, recombinant protein production was investigated in an immobilized cell culture with medium replacement. Sf9 insect cells were naturally entrapped within reticulated polyvinyl formal resin biomass support particles (BSPs; 2 mm × 2 mm × 2 mm; pore diameter 30–50 μm) in a 2.5-l stirred-tank bioreactor. The immobilized cells were grown to a density of over 10 7 cells/cm 3-BSP, with the medium of 10% fetal bovine serum (FBS)-supplemented TNM-FH replaced at appropriate intervals, before infection with a recombinant baculovirus carrying the β-galactosidase gene. When serum-free TNM-FH was used instead of 10% FBS-supplemented TNM-FH for medium replenishment on and after post-infection day 1, the immobilized cells showed a high β-galactosidase yield comparable to that obtained in a culture using the serum-supplemented medium throughout. This finding indicates that immobilized insect cell culture allows not only intensification of cell culture but also recombinant protein production in protein-free basal media.

Polyvinyl formal resin plates impregnated with lipase-entrapped sol–gel polymer for flavor ester synthesis

Polyvinyl formal (PVF) resin plate was used as a support for forming organic–inorganic hybrid sol–gel polymer within its pores. Candida cylindracea lipase immobilized by entrapment in such hybrid sol–gel polymers made from tetramethoxysilane (TMOS) and propyltrimethoxysilane (PTMS) could efficiently catalyze direct esterification reaction between geraniol and acetic acid in anhydrous hexane for synthesis of the flavor ester geranyl acetate. The optimum formulation of the sol–gel solution for enzyme activity should be made with PVF plate with 250 μm mean pore diameter, PTMS molar percentage=80%, enzyme loading at 60 mg lipase/g gel, water to silane molar ratio=25, and 4 ml PVA solution (4% w/w). The specific activity of the immobilized enzyme is 7.4 times that of the free enzyme with a protein immobilizing efficiency of 93%. The substrate inhibition by acetic acid was alleviated with the optimum acetic acid concentration, which gives the highest enzyme activity, increasing from 15 to 35 mM after enzyme immobilization. Excellent thermal stability was found for the immobilized enzyme, where full retention of enzyme activity was found after incubated at 90 °C for 2 h. When stored at room temperature, the immobilized enzyme did not show any activity loss for up to 1 month. The operational stability also increased 51-fold after entrapment in sol–gel polymer.

Immobilization of Candida Cells Showing Mycelium-Like Shapes in Porous Polyvinyl Formal Resin and Their Applications.

Candida rugosa ATCC14830 cells have high tendency to make flocculent masses by entanglement of the mycelium-like shaped cells and can be well immobilized in porous polyvinyl formal (PVF) resin. Moreover, Candida lipolytica cells, which became to show a mycelium-like shape by mutation, were also immobilized in PVF resin.These C. rugosa and mutant C. lipolytica cells were immobilized in PVF resins and cultured by the draw-fill method 2–3 times. These C. rugosa and C. lipolytica cells could decompose L-lactic acid and produce bio-surfactant liposan, respectively. Because high cell densities could be attained in immobilized systems, the total productivity in repeated-batch operations with immobilized cells was higher than those in batch operations with free cells. Moreover, the decomposed amount of L-lactic acid did not decrease during the repeated-batch operations, because the number of immobilized cells in PVF resin was kept almost constant.

A high-density culture of hepatocytes using porous substrate for use as a bioartificial liver

High-density, large-scale culture of hepatocytes is a key requirement in the development of a bioartificial liver that can replace liver functions in patients with severe liver insufficiency. We have applied a porous polymer, polyvinyl formal (PVF) resin, as a cell-supporting material for hepatocyte culture. We evaluated the performance of the culture system using PVF resin under three different culture conditions: a shake culture on conventional dishes, perfusion culture with sheet-shaped PVF, and a packed-bed-type module. Among them, the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes (2×107 cells/cm3-PVF). The hepatocytes immobilized in the PVF resin maintained satisfactory metabolic functions (ammonium metabolism and albumin secretion) comparable to those of the monolayer dish cultures. Furthermore, by maintaining dissolved oxygen concentration at a relatively high level (260–460μM), the metabolic functions of the hepatocytes were improved. It was concluded that the packed-bed reactor using PVF resin is a promising system for developing a bioartificial liver using hepatocytes.

Improvement of metabolic performance of hepatocytes cultured in vitro in a packed-bed reactor for use as a bioartificial liver.

A packed-bed reactor using reticulated polyvinyl formal (PVF) resin as a support material is a useful configuration to achieve high density culture of hepatocytes for use as a bioartificial liver. The authors investigated the effects of oxygen concentrations of the culture medium on the metabolic performance of hepatocytes cultured in the reactor. A packed-bed reactor loaded with 250 PVF resin cubes (2 x 2 x 2 mm) was used. Hepatocytes obtained from male Wistar rats were inoculated into the reactor. Culture medium was perfused from the reservoir into the reactor through an oxygenator using a roller pump. Concentration of the dissolved oxygen in the medium was controlled by changing the gas mixture ratio supplied to the oxygenator. Hepatocytes cultured in the packed-bed reactor (cell density: 8.6 x 10(6) cells/cm3 PVF) under conditions of high dissolved oxygen concentrations ranging from 260 to 460 micromol/L showed 30% higher ammonium metabolic activity and 85% higher albumin secretion activity compared with those from the monolayer culture in the earlier culture stage (up to 2 days). However, low oxygen concentrations in the medium (<100 micromol/L) impaired activities of cultured hepatocytes.

Hepatocyte culture utilizing porous polyvinyl formal resin maintains long-term stable albumin secretion activity

To investigate the effects of culture conditions on the maintenance of metabolic functions of cultured hepatocytes, long-term hepatocyte culture lasting 20 days was performed under two different culture conditions, i.e. stationary cultures utilizing porous polymer (polyvinyl formal (PVF) resin) as a substratum and conventional monolayer dish cultures without PVF. Metabolic activities specific to hepatocytes were evaluated in terms of ammonia metabolism, urea synthesis, and albumin secretion. Concerning ammonia metabolic and urea synthetic activities, no significant differences in maintenance of these activities were found between the two culture conditions, and these activities rapidly decreased with the elapse of the culture period, especially during the early stage of the experiments. However, after day 10, these activities in the stationary cultures were maintained at a slightly more favorable level than in the monolayer cultures. On the other hand, compared with ammonia metabolism and urea synthesis, stable and well-maintained albumin secretion of hepatocytes (60% of the activity in day 1) was exhibited in the stationary culture experiments, despite that this particular activity under the monolayer culture condition gradually reduced to a very low level (5.7% of that on day 1) at the end of the culture. From the morphological observations, hepatocytes immobilized in the PVF resin revealed individual spherical shapes without forming multicellular aggregation, and it was suggested that this characteristic structure contributed to good albumin secretion of hepatocytes. In conclusion, the advantages of the hepatocyte culture technique utilizing PVF resin over the conventional dish culture in maintaining some representative metabolic function specific to hepatocytes were clarified.

Packed-bed type reactor to attain high density culture of hepatocytes for use as a bioartificial liver.

In an attempt to develop a bioartificial liver using cultured hepatocytes, we investigated the short-term and long-term viability and metabolic functions of hepatocytes cultured in a new type of packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Perfusion culture experiments using this reactor, as well as monolayer cultures using conventional collagen-coated Petri dishes as control experiments, were performed. It was found that the highest density of immobilized hepatocytes attained with PVF resin was on the order of 10(7) cells/cm3 PVF and that hepatocytes cultured in this type of module for up to a week showed a sufficient level of liver-specific metabolic functions, such as ammonium metabolism, urea-N synthesis, and albumin secretion, to be comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising means to develop a bioartificial organ using hepatocytes.

Continuous IgG production by myeloma cells immobilized within porous support particles

IgG production kinetics of mouse myeloma MPC-11 cells immobilized within porous polyvinyl formal resin biomass support particles (BSPs) was evaluated by monitoring of lactate dehydrogenase activities and IgG concentration in a perfusion culture of the cells. Cells immobilized within the BSPs could stably maintain a high specific IgG production rate, comparable to the maximum production rate in a static culture of non-immobilized MPC-11 cells, over a long period.

Long-term performance of albumin secretion of hepatocytes cultured in a packed-bed reactor utilizing porous resin.

A packed-bed reactor utilizing the porous polymer, polyvinyl formal (PVF) resin, as a supporting material was applied for the long-term culture of rat hepatocytes. Perfusion cultures using this reactor, as well as monolayer cultures, were performed for up to 1 week under 3 different media conditions. When serum-free or epidermal growth factor-containing medium was used, albumin secretion rates showed constant decreases in both the perfusion and the monolayer cultures, and approximately 10% of the activity exhibited on Day 1 was preserved at the end of the cultures. In contrast, the hepatocytes supplemented with serum-containing medium exhibited stable ability of albumin secretion throughout the culture period under the perfusion culture condition using PVF resin. From the microscopic observation, the immobilized hepatocytes in this medium revealed round shapes, and a cluster of cell aggregates was scarcely seen.

Long-term cultures of hepatocytes using reticulated polyvinyl formal resin as a substratum

Long-term cultures of hepatocytes using reticulated polyvinyl formal resin as a substratum

Growth kinetics of animal cells immobilized within porous support particles in a perfusion culture

The growth kinetics of anchorage-independent animal cells [mouse myeloma MPC-11 (ATCC CCL 167)] immobilized within porous polyvinyl formal resin biomass support particles (BSPs; 3 x 3 x 3 mm cubes; mean pore diameter, 60 microns) was analyzed by measuring intracellular and extracellular lactate dehydrogenase (LDH) activities in a perfusion culture. First, the intracellular LDH content of cells immobilized within the BSPs was assayed in a shake-flask culture and found to remain almost comparable to that of non-immobilized cells in the exponential growth phase under static culture. Then, cells inoculated in the BSPs were grown in a continuous stirred-tank bioreactor. Using the LDH content of non-immobilized cells, the density of immobilized cells and the numbers of leaked and dead cells were evaluated, respectively, by the intracellular LDH activity of immobilized and leaked cells and the LDH activity in cell-free culture supernatant. In the initial period, immobilized cells exhibited exponential growth at a constant apparent specific growth rate; however, the actual specific growth rate, which takes into consideration cell death and cell leakage, decreased significantly. In the stationary phase, the actual specific growth rate maintained a constant but markedly lower value than during exponential growth.

Long‐Term continuous culture of hepatocytes in a packed‐bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.

Coculture of hepatocytes using polyvinyl formal resin with two kinds of nonparenchymal cells

Coculture of hepatocytes using polyvinyl formal resin with two kinds of nonparenchymal cells

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Growth and death behaviour of anchorage-independent animal cells immobilized within porous support matrices.

Growth and death of anchorage-independent animal cells entrapped within porous biomass support particles (BSPs) in static or shake-flask cultures were evaluated by comparison of enzyme activity with non-immobilized cells grown under static culture using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and release of lactate dehydrogenase into the culture medium. Mouse myeloma MPC-11 (ATCC CCL 167) cells inoculated within porous polyvinyl formal resin BSPs (3 x 3 x 3 or 2 x 2 x 2 mm; mean pore diameter, 60 microns) grew exponentially at a specific growth rate comparable to that of non-immobilized cells in the initial period of incubation. Entrapped cells then reached the stationary phase with a cell density over 10(7) cells/cm3 BSP. The death rate of entrapped cells increased in response to the rise in viable cell density in the BSPs. Observation of viable cell distribution within the BSPs using MTT staining indicated that the cells concentrated within a thin outer shell of the BSPs with time. After the immobilized cells reached the stationary phase, penetration of cells into the outer shell ceased and heterogeneous distribution of cell density occurred in the viable cell layer in the shake-flask culture.

A packed-bed reactor using reticulated polyvinyl formal resin

A packed-bed reactor using reticulated polyvinyl formal resin