Chemically-defined Serum-free Medium Research Articles

Abstract 3073: Bladder cancer patient-derived organoids to decipher cellular plasticity and cancer progression

Abstract Introduction: Muscular Invasive Bladder Cancer (MIBC) is a highly heterogeneous disease. A consensus classification, based on gene expression profiling, has identified six MIBC subgroups, ranging from basal/squamous (Ba/Sq) to luminal papillary (LumP). This classification is a static representation of the disease and does not account for cellular plasticity, which may induce phenotype switch and cancer progression. Here, we aimed at identifying factors involved in cellular plasticity using bladder cancer patient-derived organoids (PDO) models. Methods: Bladder cancer RNA sequencing data from bulk and single cell experiments were downloaded from public repositories. Samples from both datasets were classified using the consensus molecular classification. Pathway activation scores and cell-cell interaction scores were calculated for each subtype. Differential gene expression analysis was performed with the R packages edgeR and Seurat. MIBC samples were collected from bladder cancer patients undergoing surgical treatment. The samples were mechanically and enzymatically digested to generate a cell suspension which was seeded in Matrigel and immersed in growth-factor supplemented medium. Phenotypical characterization of long-term PDO lines was performed through immunofluorescence (IF). The PDOs were classified as Luminal, Basal or Mixed, based on the number of cells expressing the luminal marker CK8 or the basal marker CK5. Three selected organoids lines were cultured in alginate, immersed in a serum-free chemically-defined medium. Results: Long-term PDO lines were successfully generated from 6 out of 13 MIBC samples. Organoids from the same line showed a consistent expression of basal and luminal markers. One line was classified as Basal, 2 lines as Mixed and 3 lines as Luminal. Exploring bulk RNAseq and scRNAseq datasets, we identified several genes whose expression was significantly altered in Ba/Sq and LumP MIBC subtypes. In particular, SHH and related BMP genes were significantly less expressed in Ba/Sq samples than in LumP. In addition, we observed an overexpression of BMP3 in LumP samples and of TGFβ2 in Ba/Sq samples. As preliminary test, we formulated a chemically-defined medium containing inhibitors of the proteins encoded by the identified genes (TGFβ inhibitor and Noggin); using this medium, we were able to maintain organoids from a Luminal, Basal and Mixed subtype in culture for up to two weeks. Interestingly, after two weeks of culture, IF analysis highlighted the presence of cells expressing luminal markers in the basal organoid line, suggesting culture-induced change of phenotype. Conclusions: Bladder cancer PDOs represent promising models to study MIBC plasticity. The integration of RNA sequencing data and the successful culture of PDOs in chemically-defined culture conditions may allow to identify candidate factors impacting the phenotype of bladder cancer cells. Citation Format: Michele Garioni, Luca Roma, Renaud Mével, Tatjana Vlajnic, Heike Pueschel, Dagmar Iber, Hans-Helge Seifert, Cyrill A. Rentsch, Lukas Bubendorf, Clémentine Le Magnen. Bladder cancer patient-derived organoids to decipher cellular plasticity and cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3073.

Platform for the growth and propogation of HEK293 cells and adenovirus viral vector amplification

Background & Aim Industrialization of viral vector production for gene therapy applications demands consistent processes utilizing robust production platforms and raw materials that can meet quality standards and regulatory compliance. Typical vector production schemes involve propagating seed trains and viral amplification in cultures utilizing serum containing media. Although serum-containing media systems deliver high viral vector yields, the serum component has certain disadvantages such as performance variability, high costs, and potential of introducing adventitious agents. Utilizing serum-free chemically-defined medium for growth and amplification addresses these concerns. Here we describe a highly efficient and economical single-use bioreactor platform for producing gene therapy viral vectors with flexibility of using serum-containing or serum-free media. Methods, Results & Conclusion We evaluated multiple serum-free chemically-defined media formulations for adenovirus vector production in the single-use fixed-bed iCELLis® bioreactor. These media were evaluated for ease of adaptation and growth characteristics with HEK293 cells previously adapted to serum-free growth conditions. Process parameters such as multiplicity of infection (MOI) and duration of infection (DOI) were optimized in a shake flask to maximize vector amplification. The process was then transferred into the iCELLis Nano bioreactor. The HEK293 cells were maintained under adherent conditions and growth was monitored until the cell densities reached approximately 200,000 cells/cm2. Complete medium exchange was performed in iCELLis Nano bioreactor without cell removal, followed by viral infection at MOI 50. Harvest was performed 72 hours post infection. Viral titers were quantitated using an adenovirus in-vitro infectivity assay. Viral productivity was generally consistent between the shake flask and the iCELLis Nano bioreactor system and between serum-containing or serum-free chemically-defined medium. These studies demonstrate the iCELLis bioreactor as a versatile scalable technology, and support its use for production of gene therapy vectors in processes using serum-containing or serum-free media.

The effects of insulin, transferrin and androgens on rat prostate explants in serum-free organ culture.

A model previously developed in our laboratory to culture rat prostate explants in serum-free chemically-defined medium was used to evaluate the direct influence of potential regulators. The aim of the present work was to verify the effects of insulin (I) and transferrin (Tr), two hormones considered as essential in other serum-free culture systems, and three androgenic hormones, since the prostate is known to be androgen-dependent. Explants of rat prostate were cultured for five days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air-5% CO2). The addition of Tr (50 micrograms/ml) had no effect, but I (5 micrograms/ml) significantly increased DNA synthesis. This influence was amplified by combination of the two hormones. However, protein synthesis was only slightly stimulated. Testosterone (T) or androstanediol significantly increased DNA synthesis when compared to corresponding control values at five days. In combination with I plus Tr, each hormone showed potentiated effects, particularly T with a twofold increase over day 0 values. When dihydrotestosterone was added singly, the incorporation of 3H-thymidine was stimulated by 300% over control values at five days, and by 100% over values in uncultured explants. This influence was maximal since it was not improved by I plus Tr. Protein synthesis was increased significantly by the triple combination. In addition, each androgen as well as the combination of I plus Tr had a positive influence on explant morphology. The above conditions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors in prostate metabolism and to eventually identify putative cancer markers.

器官培養法を用いたマウス口蓋突起癒合期のビタミンの効果に関する実験的研究

I studied the effects of vitamins on the prevention of cleft palate in A/J mice with the use of an organ culture suspension. Maxillary regions of A/J mouse fetuses (13.5 days) were dissected and cultured in a chemically-defined serum-free medium for 72h.Each vitamin (β-carotene, vitamin E, folic acid) was added to the medium in concentrations of 0.1, 1.0, and 10μ g/ml.In the β-carotene groups, the rates of palate fusion were 74.1%(control), 76.2%(0.1μg/ml), 90.0%(1.0μg/ml), and 94.1%(10μg/ml).In the vitamin E groups, the rates of palate fusion were 53.0%(control), 63.6%(0.1μg/ml), 71.4%(1.0μg/ml), and 80.0%(10μg/ml).In the folic acid groups, the rates of palate fusion were 60.0%(control), 85.7%(0.1μg/ml), 85.2%(1.0μg/ml), and 100%(10μg/ml).Significant differences from control were obtained when folic acid was added to the medium in concentrations of 0.1, 1.0, and 10μg/ml.These results indicate that vitamins, in particular folic acid, have a direct action on the palatal shelves.Although the mechanism of the effect of vitamins is presently unclear, these results may be useful medically in the prevention of cleft lip, cleft palate, or both.

EFFECTS OF ETHANOL ON FETAL BRAIN REAGGREGATE CULTURES. † 469

Fetal alcohol syndrome is the leading known cause of mental retardation. Clinical and experimental studies show a number of specific biochemical and morphological abnormalities of the cerebral cortex following ethanol exposure. To further characterize the direct effects of ethanol on fetal brain development, we have utilized a fetal brain reaggregate cell culture system grown in a serum free, chemically defined medium (SFCD). Cerebral cortices from gestational day 16 Sprague-Dawley fetal rats were removed. The brains were dispersed into single cells by mechanical dissociation, plated into SFCD media, and placed on a gyratory shaker in 10% CO2 at 37°C. After 48 hours, ethanol was added for a final concentration of 32 mM. Oxygen consumption, DNA and protein content, and DNA synthesis (thymidine uptake) were measured as a function of time. The results are shown in the followingtable (Ctl-control, EtOH-ethanol):

A clone of the MOCH-1 glial tumor in culture: multiple phenotypes expressed under different environmental conditions.

The MOCH-1 glial cell line, which was derived from a glioblastoma taken from the brain of a MBP/c-neu transgenic mouse, was used as a model for studying the plastic nature of gliomas in culture. Fifteen MOCH-1 clones were derived and characterized under different growth conditions via Western blot analysis and immunocytochemical staining using a panel of antibodies specific for major myelin markers and glial fibrillary acidic protein. In low serum conditions, the clones resemble immature oligodendrocytes and express only immature oligodendrocyte markers. When placed in serum-free chemically defined medium (CDM), nine clones do not change their phenotypes, while five variably express myelin markers. One clone, 4C8, differentiates into mature, membrane sheet-bearing oligodendrocyte-like cells and expresses major myelin markers in amounts and distributions similar to cultured primary oligodendrocytes. Our data show that 4C8 can reversibly switch from an oligodendrocyte-like phenotype to a reactive astrocyte-like phenotype, depending upon the presence of serum and other factors in different growth media. Our data indicate that serum "pushes" 4C8 into the astrocyte-like phenotype, while serum-free CDM "pushes" 4C8 into the oligodendrocyte-like phenotype. Furthermore, a population of mixed phenotypes results when the astrocyte-like 4C8 cells are placed in serum-free CDM. To our knowledge, this is the first report to show that by altering environment, a "mixed glioma" can arise from a homogeneous population of glial tumor cells. It is therefore possible that glial tumor cells in vivo may be induced to undergo similar phenotypic changes when they are exposed to different environmental signals in the central nervous system.

Cellular Toxicity of X-ray Contrast Media for Two Renal Epithelial Cell Lines (MDCK and LLC-PK1)

The two renal cell lines, MDCK (distal tubule) and LLC-PK1 (proximal tubule), have been used for toxicity testing of three different X-ray contrast media: the ionic monomer Isopaque, the ionic dimer Hexabrix and the non-ionic monomer Omnipaque. The cells were grown to confluency in monolayer cultures in a chemically-defined serum-free medium before the contrast media were added, to give final concentrations corresponding to 0–100mg iodine/ml. Toxicity was assessed by cell viability and by biochemical assays of marker enzymes. The results demonstrate a concentration-dependent toxic effect from the contrast media on cellular appearance, and on the activity of brush border and lysosomal enzymes. The non-ionic X-ray contrast media appeared to be less toxic than the ionic contrast media investigated.

Expression of liver-specific functions and secretion of a hepatocyte growth factor by a newly established rat hepatoma cell line growing in a chemically-defined serum-free medium

A rat hepatoma cell line 3'-mRLh-2 was established from 3'-methyl-4-dimethylaminoazobenzene-induced hepatoma. Cells proliferated well in 5Fs-DM-160, a chemically-defined serum-free medium; population doubling time was 68.5 h, and modal chromosome number was 81 (21%). The cells were transplantable, and the transplanted tumors were histologically diagnosed as hepatocellular carcinoma. They were cytochemically positive for gamma-glutamyl transpeptidase. The cells possessed about 30% of tyrosine aminotransferase activity level in the rat liver, and showed 5.5 to 7.4-fold induction of this enzyme activity in response to dexamethasone. Also, the cells secreted alpha-fetoprotein, albumin, transferrin, alpha 1-acid glycoprotein, C-reactive protein, fibrinogen, complement component C3, and other five-serum proteins. Furthermore, the conditioned medium stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. These properties of the cell line 3'-mRLh-2 were compared with those of the popular rat hepatoma cell lines, such as H4-II-E-C3 from Reuber hepatoma H35 and HTC from Morris hepatoma 7288C.

Mucin production by colon cancer cells cultured in serum‐free medium

Although many colon cancer cell lines are available for study, few of them exhibit differentiated properties. When cultured in medium containing fetal bovine serum, WiDr cells (WiDr-FBS) show an undifferentiated phenotype: growth as a multilayer of cells adherent to plastic and lack of polarization, brush border, and mucin vacuoles. In contrast, WiDr cells cultured in a chemically-defined serum-free medium containing insulin, transferrin and selenium (WiDr-ITS) grow as clusters of nonadherent cells with abundant desmosomes and tight junctions, microvilli and electron-lucid vacuoles. As WiDr-FBS cells, WiDr-ITS are not polarized. WiDr-ITS cells show a marked enhancement in mucin synthesis as demonstrated by: periodic acid-Schiff and Alcian blue stains, electron microscopy, immunohistochemistry using monoclonal antibodies (MAbs) reactive with mucin-associated epitopes, immune electron microscopy and immunochemical analysis using Western blots. In comparison with WiDr-FBS cells, WiDr-ITS cells showed strong expression of Tn, sialyl-Tn, blood group A and CEA. When mouse MAbs were used, higher levels of the MUCI gene product were detected in WiDr-ITS than in WiDr-FBS cells. The full spectrum of phenotypic changes was observed after I month of culture in ITS medium, and transfer of WiDr-ITS cells to FBS medium was accompanied by a partial phenotypic reversal, suggesting that these phenotypic changes result from an adaptative--rather than selective--process.

A Comparison of Neurotrophic Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor in Primary Cultured Neurons from Various Regions of Fetal Rat Brain

Effects of recombinant human epidermal growth factor (hEGF) on survival of primary cultured neurons from various regions of fetal rat brain were compared with those of recombinant human basic fibroblast growth factor (hbFGF). Addition of hEGF (0.001-10 ng/ml) in chemically-defined serum-free medium enhanced significantly the survival of cultured neurons from all regions tested, i.e., cerebral cortex, septum, striatum, hippocampus, thalamus, colliculus and cerebellum. However, its effective concentration differed considerably with the regions. The most sensitive regions were the colliculus and cerebellum (minimum effective concentration, 0.001 ng/ml), while the least sensitive was the cerebral cortex (minimum effective concentration, 10 ng/ml). Maximum numbers of surviving neurons in the presence of hEGF were smaller than those of hbFGF in all regions. Maximum effects of hEGF compared with those of hbFGF was greatest in the striatum and smallest in the cerebral cortex. These results suggest that hEGF may exert neurotrophic effects for limited populations of brain neurons. When hEGF (1-10 ng/ml) and hbFGF (0.01-10 ng/ml) were added together, hEGF increased the effects of a lower concentration of hbFGF in an additive manner, but the maximum effects of hbFGF were not potentiated by hEGF in all regions tested. Therefore, mechanisms underlying the effects of hEGF very probably overlap with those of hbFGF.

1,25-Dihydroxyvitamin D 3-induced growth restriction of cultured epithelial cells derived from a murine hepatic tumor

Recent evidence suggests that 1,25-dihydroxyvitamin D 3 can accumulate in certain presumed non-target tissues, although the mechanism of action of the vitamin in such cells is not understood. Exposure of 77-1/3a mouse hepatic tumor cells, which derived from a non-target tissue of vitamin D action, to 1,25-dihydroxyvitamin D 3 in chemically-defined serum-free medium resulted in a dose-dependent decline in cellular growth rate and maximal culture population density but did not adversely affect cell viability. Culture of 77-1/3a cells in defined medium containing 10 −7 or 10 −6M 1,25-dihydroxyvitamin D 3 for 150 hr reduced the growth rate to 64 and 50% of control values respectively. Albumin secretion was unaffected by 1,25-dihydroxyvitamin D 3 exposure; in contrast, the cellular content of the proliferation-associated protein p35 was reduced by 39%, a decline similar in trend and degree to that observed in other tumor cells exposed to differentiation-inducing agents. It appears that 1,25-dihydroxyvitamin D 3 regulates cellular p35 content (within a specific restricted range) as a consequence of proliferative perturbation, rather than differentiated status, of cultured hepatic tumor cells.

Effects of insulin and insulin-like growth factor (IGF-I) on oligodendrocyte-enriched glial cultures.

The brains from 1-week-old rat pups were used to prepare cultures of glial cells. After 24 hr in culture the cells were changed to a chemically defined serum-free medium (CDM). We have used antibodies against gangliosides (monoclonals A2B5 and LB1) and against galactocerebrosides to monitor the influence of insulin on the development of oligodendrocytes from precursor cells. In these oligodendrocyte-enriched cultures we investigated the influence of insulin and IGF-I on the activity of the marker enzymes 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and glycerol-3-phosphate dehydrogenase (G3PDH), and on lipid metabolism. 1) Incorporation of [35S] sulfate into sulfolipids was stimulated by insulin (optimal concentration 100 nM). A sharp peak in sulfolipid synthesis was seen at day 5-6 in culture. 2) Insulin stimulated the expression of CNPase and G3PDH in culture. 3) The stimulating effect of insulin on sulfolipid synthesis, CNPase, and G3PDH activity was mimicked by IGF-I (13 nM). 4) The incorporation of [35S] into sulfolipids and [2-3H]acetate into fatty acids and cholesterol was reversibly reduced by temporary omission of insulin from the medium. These results indicate that insulin and IGF-I stimulate the development of oligodendrocytes in our culture system and that insulin has a general anabolic effect on the lipid metabolism of the cells.

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D 3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 mg/ml) allowed a more precise examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10 −10 to 10 −8 M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants ( K d ) nor in the number of calcitriol binding sites per HL-60 cell, that is ~6.0 × 10 −11 M and ~3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/ or unidentified components present in serum.

Effects of chemical additives on functional innervation patterns in mouse spinal cord-ganglion explants in serum-free medium

The distribution of sensory evoked bioelectric activity was examined in spinal cord-dorsal root ganglion (SC-DRG) preparations cultured in a chemically defined, serum-free medium (CDM). DRG afferents showed no preferential innervation of dorsal cord regions in this CDM, on the basis of electrophysiological mapping of the distribution of evoked responses at 4 weeks in vitro. Addition of chondroitin sulfate, galactose-1-phosphate or d(+)-galactose (but not glucose-1-phosphate) to the CDM resulted in a significant increase in presumed monosynaptic connections within the dorsal cord, thus mimicking the results observed in serum-supplemented medium [1,6]. Inasmuch as d(+)-galactose bears no negative charges yet restores the selective functional innervation, whereas glucose-1-phosphate (a highly charged molecule) fails to do so, it is concluded that it is galactose utilization, rather than the charged nature of the chondroitin sulfate and galactose-1-phosphate molecules, which is responsible for the effect.

Nerve growth factor-mediated differentiation of a nerve cell line cultured in a hormone-supplemented serum-free medium.

Abstract In newly-isolated subclone of PC12, designated h, nerve growth factor (NGF) is known to cause an increase of tyrosine hydroxylase (TH) activity concomitantly with that of choline acetyltransferase (CAT) activity16. When the PC12h cells were cultured and maintained for several generations in a hormone-supplemented serum-free medium, only TH activity was selectively enhanced by NGF, while CAT and glutamic acid decarboxylase activities remained unaffected. PC12h cells cultured in serum-free medium could extend long neurites in response to NGF. The loss of the NGF-mediated increase of CAT activity in PC12h cells cultured in serum-free medium was fully restored upon re-exposure to serum. The NGF-mediated increase of TH activity in PC12h cells cultured in serum-free medium was additive to that increased by an applications of 10−5-10−10 M dexamethasone. In conclusion, PC12h cells cultured in chemically-defined serum-free medium, having responses to NGF identical to sympathetic neurons, will be a useful model for elucidating the molecular mechanism(s) of NGF-mediated neuronal differentiation.

Evidence for the retinoid control of urothelial alkaline phosphatase

The amount of alkaline phosphatase activity per μg of DNA in the urothelium (transitional epithelium) of the rat urinary bladder, organ-cultured in chemically-defined serum-free medium, decreased greater than 70% during a 13 day culture period. This decrease in enzyme activity corresponded inversely with the increase in cell number in the urothelium indicating that enzyme synthesis did not accompany growth. Alkaline phosphatase activity was increased back to values approaching normal enzyme levels during a 3 day culture period by the addition of 10 μM retinoic acid. Retinol also increased enzyme activity but it was only half as effective as retinoic acid. A significant increase in enzyme activity was initiated by 1 μM retinoic acid, however the most effective concentration was at 10 μM.

Induction of epidermal transglutaminase by hydrocortisone in chick embryonic skin.

WE demonstrated previously that the epidermal cells of chick embryos are induced to differentiate by glucocorticoids1,2. Hydrocortisone (0.01 µg ml−1) added to a serum-free chemically-defined medium produced an intense keratinised layer over the uppermost cells3 and many tonofilaments within the basal and intermediate cells4 of the epidermis after 4 d in culture of 13-d-old embryonic chick tarsometatarsal skin. Within 12–16 h of hydrocortisone addition it is possible to show the synthesis of a urea–mercaptoethanol soluble, glycine-rich and metabolically stable epidermal structural protein5 and the accumulation of a urea–mercaptoethanol insoluble epidermal protein(s)4. These changes of protein metabolism induced by hydrocortisone paralleled those seen during in ovo keratinisation of the epidermis4,5. The amino acid composition of the two proteins was very similar suggesting that the soluble protein is converted to the insoluble one in the process of epidermal keratinisation of chick embryonic skin. We show here that the levels of transglutaminase (TG), an enzyme that possibly mediates cross-linking of keratinous polypeptide chains by a ɛ-(γ-glutamyl) lysine bond6–8, increases both during in ovo development of the epidermis and during hydrocortisone-induced in vitro keratinisation of chick embryonic cultured skin.