Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

Observations of an extreme form of late eccentric lytic fission in Schizosaccharomyces pombe

Abstract Late eccentric lytic fission was initially observed in Schizosaccharomyces pombe in 1967 (Johnson, B.F. (1967) J. Bacteriol. 94, 192–195). We report here an extreme form of this morphology and have studied the incidence of severity with respect to glucose concentration of the growth media. Alterations to the composition of the growth medium were made to allow cells to reach a density of 1 × 10⁸ cells ml⁻¹ before lysis occurred.     

Effect of Tween 80 on glucosyltransferase production in Streptococcus mutans.

Glucan production from sucrose by Streptococcus mutans OMZ 176 was stimulated approximately threefold in the presence of 0.1% Tween 80. When OMZ 176 was grown in a medium containing glucose, the glucosyltransferase level in the medium was also increased about fivefold in the presence of 0.1% Tween 80. The glucosyltransferase level increased in proportion to the logarithm of the concentration of Tween 80 in the glucose medium. Tween 80 affected neither bacterial growth nor the activity of glucosyltransferase. The appearance of glucosyltransferase in the glucose medium was inhibited immediately by chloramphenicol and actinomycin D and, after a lag, by rifampin as well. It was observed that the fatty acid composition of the cells grown with Tween 80 was altered. These results suggest that Tween 80 stimulates glucosyltransferase synthesis either directly, or indirectly by promoting glucosyltransferase secretion.     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

Hexose uptake and control of fibroblast proliferation

The role of glucose uptake in control of cell growth was studied by experimentally varying the rate of glucose uptake and examining the subsequent effect on initiation and cessation of cell proliferation. The rate of glucose uptake was varied by adjusting the concentration of glucose in the culture medium. This permitted analysis of two changes in rate of glucose uptake which are closely related to the regulation of cell growth: (1) the rapid increase in glucose uptake that can be detected within several minutes after mitogenic stimulation of quiescent fibroblasts and (2) the decrease in glucose uptake which accompanies growth to a quiescent state. Quiescent cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to fresh serum-containing medium with either the normal amount of glucose or a reduced level that lowered the rate of glucose uptake below the rate characteristic of quiescent control cells. The subsequent increases in cell number were equal in both media, demonstrating that the increase in glucose uptake, commonly observed after mitogenic stimulation, was not necessary for initiation of cell division. Measurements of intracellular D-glucose pools after serum stimulation of quiescent cells revealed that the increase in glucose uptake was not accompanied by a detectable change in the intracellular concentration of glucose. Nonconfluent growing cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to low glucose media, lowering the rate of glucose uptake below levels observed for quiescent cells. This did not affect rates of DNA synthesis or cell division over a several-day period. Thus, the decrease in glucose uptake, which usually occurs at about the same time as the decrease in DNA synthesis as cells grow to quiescence, does not cause the decline in cell proliferation. Experiments indicated that there was no set temporal relationship between the decline in glucose uptake and DNA synthesis as cells grew to quiescence. The sequence was variable and probably depended on the cell type as well as culture conditions. Measurements of intracellular D-glucose pools in secondary chick embryo cells demonstrated that the internal concentration of glucose in these cells did not significantly vary during growth to quiescence. Taken together, our results show that these fluctuations in the rate of glucose uptake do not lead to detectable changes in the intracellular concentration of glucose and that they do not control cell proliferation rates under usual culture conditions.     

Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.     

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: The nature of the signal

Summary We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK₁. Assayed in physiological salt solution, the ratio of the concentration of -methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK₁ cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1mm glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.     

Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector

PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 x 10(6) cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 x 10(6) cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation.     

Some aspects of hybridoma cell cultivation

Summary Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injection analyzer and the ammonia concentration by an ion-selective electrode. Since the membrane transmission of the high-molecular components decreased during cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and consumption rates of the medium components were influenced considerably by the medium composition, especially by the type and amount of serum used.Offprint requests to: K. Schügerl     

Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve

Marine sponge cell culture is a potential route for the sustainable production of sponge-derived bioproducts. Development of a basal culture medium is a prerequisite for the attachment, spreading, and growth of sponge cells in vitro. With the limited knowledge available on nutrient requirements for sponge cells, a series of statistical experimental designs has been employed to screen and optimize the critical nutrient components including inorganic salts (ferric ion, zinc ion, silicate, and NaCl), amino acids (glycine, glutamine, and aspartic acid), sugars (glucose, sorbitol, and sodium pyruvate), vitamin C, and mammalian cell medium (DMEM and RPMI 1640) using MTT assay in 96-well plates. The marine sponge Hymeniacidon perleve was used as a model system. Plackett-Burman design was used for the initial screening, which identified the significant factors of ferric ion, NaCl, and vitamin C. These three factors were selected for further optimization by Uniform Design and Response Surface Methodology (RSM), respectively. A basal medium was finally established, which supported an over 100% increase in viability of sponge cells.     

Effect of Type of Carbohydrate on Growth and Protein Synthesis by Tetrahymena pyriformis

SYNOPSIS. In chemically defined media at carbohydrate concentrations ≧ 0.5% (w/v) Tetrahymena pyriformis W multiplied more rapidly, developed larger cells, and achieved greater growth as measured by optical density when carbohydrate was provided as dextrin rather than glucose. In media containing 0.3 mg/ml of amino acid nitrogen, growth increased with glucose concentration from 0.1 to 1%, did not change significantly to 3%, and was sharply inhibited at higher glucose levels. With dextrin, maximum growth paralleled carbohydrate concentration from 0.1 to 3%. At higher N levels the inhibitory concentration of glucose was lowered, but growth in dextrin media was not affected except at N concentrations that were inhibitory independent of carbohydrate source. At 1% carbohydrate levels, total cell protein per ml of culture was 60% greater, protein per cell approximately 50% greater, and cells were 1.5 to 2 times larger in media with dextrin than with glucose. Comparable differences in protein synthesis were observed at 2% carbohydrate levels and efficiency of conversion of substrate-N to protein-N was greater in the medium with dextrin than glucose.
Growth as measured by optical density in media with 0.3 mg/ml of N and 1 or 2% (w/v) of dextrin was not significantly reduced by the simultaneous presence of 1 or 2% glucose. This observation appeared to negate osmotic pressure as an explanation of reduced growth in the presence of glucose. At higher osmolar concentrations osmotic pressure appeared to be a major determinant of overall growth but not of cell size.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Growth behavior of Chinese hamster ovary cells in a compact loop bioreactor. 2. Effects of medium components and waste products.

Effects of biochemical factors, i.e., medium components and metabolic byproducts, on growth of Chinese hamster ovary (CHO) cells were investigated. Glucose and ammonia were found to inhibit the growth. Kinetic analysis gave the inhibition constants, 0.14 g l-1 for ammonia and 5.0 g l-1 for glucose. Since glutamine was unstable and was a main source of ammonia, precise studies on glutamine degradation and ammonia formation process were done. By evaluating the spontaneous reactions, net glutamine utilization and net ammonia production by the cells could be estimated. It became evident that asparagine could support the growth of CHO cells as a stable substitute for glutamine. Then, a glucose fed-batch culture was grown on a glutamine free and asparagine supplemented medium. Because of (1) low glucose concentration, but (2) no glucose limitation and (3) low ammonia accumulation, the maximum total cell concentration reached 3.4 x 10(6) ml-1, which was 1.8 times greater than that in the control experiment (initial 1.15 g l-1 glucose and 0.29 g l-1 glutamine, and no glucose feed).     

Regulation of glucose and maltose transport in strains ofSaccharomyces

Summary Growth of yeast cells on glucose resulted in complete inactivation of maltose transport and repression of the high affinity glucose transport system. When the cells were grown on maltose or subjected to substrate starvation, an increase in glucose and maltose transport was observed in both brewing and non-brewing yeast strains. The concentration of glucose employed in the growth medium was also observed to affect sugar transport activity. The higher the glucose concentration, the more pronounced the repressive effect. In addition, the time of growth of yeast on glucose or maltose also intermining the rate of sugar transport. These results are consistent with the repressive effect of glucose on the high affinity glucose and maltose transport systems.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

Summary The effect of medium composition and initial glucose concentration on production of hEGF by recombinant E. coli cells was investigated. Optimum hEGF production was observed in a yeast extract/acid hydrolysed casein/salts media containing an initial glucose concentration of 10 g.l^-1. A maximum hEGF titer of 250 mg.l^-1 was obtained in this medium after 32 h in laboratory fermenters with pH, temperature, agitation and aeration set at 6.8, 30°C, 500 rpm and 2 vvm, respectively.     

Effects of hepatocyte growth factor and platelet-derived growth factor on the repair of meniscal defects in vitro

Summary Injuries to the avascular region of the meniscus occur frequently and may be difficult to repair. This study was designed to determine whether growth factors could diffuse from a collagen sponge or a collagen gel into meniscal tissue and stimulate healing of defects using an in vitro model. The diffusion of platelet-derived growth factor (PDGF) from the collagen carriers into the medium was rapid with approximately 50% being released from the collagen sponge within the first hour. After 5 d of incubation, 8% of the PDGF was present in the meniscus, 11% in the collagen sponge, and 62% had been released into the medium. Similar results were obtained when a collagen gel was used as a carrier. Histological evaluation of the meniscal explants after 2 wk in culture revealed extensive proteoglycan staining in the areas surrounding defects treated with either hepatocyte growth factor (HGF) or PDGF compared with controls without growth factor. The HGF-PDGF treatment resulted in alignment and migration of meniscal cells toward the defect, which was not observed in untreated controls. At 3–7 d, increased number of cells were observed in defects treated with collagen gels (but not the sponge) with PDGF-HGF. At 4 wk, combined HGF-PDGF treatment resulted in the formation of tissue with birefringence by polarized microscopy, suggestive of organized collagen. The data suggest that use of specific PDGF-HGF may enhance the repair of meniscal injuries.