Cell Culture Nutrient Research Articles

Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration

ObjectiveTissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. MethodshPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. ResultshPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. ConclusionsThe 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.

Mass Spectrometry Based Subcellular Coenzyme Analysis

Metabolism in eukaryotes relies on compartmentalization of processes between sub‐cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub‐cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture ‐ Subcellular Fractionation (SILEC‐SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13C,15N‐label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography‐high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.

Quantitative Sub-Cellular Acyl-Coa Analysis Reveals Distinct Nuclear Regulation

Quantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes, but are subject to multiple confounding factors. We developed Stable Isotope Labeling of Essential nutrients in cell Culture - Sub-cellular Fractionation (SILEC-SF), which uses isotope labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-Coenzyme A thioesters in sub-cellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing we identified the branched chain amino acid (BCAA) isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells.

Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining. Conclusion: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

Ceramic Hollow Fibre Constructs for Continuous Perfusion and Cell Harvest from 3D Hematopoietic Organoids.

Tissue vasculature efficiently distributes nutrients, removes metabolites, and possesses selective cellular permeability for tissue growth and function. Engineered tissue models have been limited by small volumes, low cell densities, and invasive cell extraction due to ineffective nutrient diffusion and cell-biomaterial attachment. Herein, we describe the fabrication and testing of ceramic hollow fibre membranes (HFs) able to separate red blood cells (RBCs) and mononuclear cells (MNCs) and be incorporated into 3D tissue models to improve nutrient and metabolite exchange. These HFs filtered RBCs from human umbilical cord blood (CB) suspensions of 20% RBCs to produce 90% RBC filtrate suspensions. When incorporated within 5 mL of 3D collagen-coated polyurethane porous scaffold, medium-perfused HFs maintained nontoxic glucose, lactate, pH levels, and higher cell densities over 21 days of culture in comparison to nonperfused 0.125 mL scaffolds. This hollow fibre bioreactor (HFBR) required a smaller per-cell medium requirement and operated at cell densities > 10-fold higher than current 2D methods whilst allowing for continuous cell harvest through HFs. Herein, we propose HFs to improve 3D cell culture nutrient and metabolite diffusion, increase culture volume and cell density, and continuously harvest products for translational cell therapy biomanufacturing protocols.

Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [13C315N1]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [13C315N1]-nicotinamide and [13C11]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).

Parathyroid Autotransplantation During Thyroid Surgery: A Novel Technique Using a Cell Culture Nutrient Solution.

Parathyroid autotransplantation is an easy procedure with a low complication rate. We adopted the transplantation into the sternocleidomastoid muscle, which allows an easier and time-saving surgical procedure using the same surgical incision. In this study, we retrospectively reviewed the records of 396 consecutive patients, who underwent total thyroidectomy for benign thyroid disease. In all cases in which a parathyroid was damaged or inadvertently removed, the gland was transplanted; before the autotransplantation, the parathyroid tissue was put in a cell culture nutrient solution for 5min, afterward fragmented, and then was transplanted in the sternocleidomastoid muscle. To demonstrate a beneficial effect of the cell nutrient solution step, we compared data of transplanted patients with a control group of cases (n=190) undergoing a standard immediate autotransplantation. We divided patients in two main groups: group A (n=160) including subjects that underwent one or more parathyroid gland autotransplantation using the cell nutrient solution, and group B (n=236) concerning those who were not transplanted. Among patients, 62 hypocalcemias occurred, 40 in the group A and 22 in the group B (P<0.001): 91.9% were transient and 8.1% (5 patients) definitive, all pertaining to the group B. Among controls (group C), 42 hypocalcemias occurred (P=0.616 vs. group A and P=0.002 vs. group B) and 3/42 became definitive (P=0.096 vs. group A and P=0.121 vs. group B). All differences concerning pre- and postoperative calcium values were statistically significant (P<0.001). We recommend the routine parathyroid autotransplantation, when a vascular damage is certain or suspected, in order to reduce the rate of permanent hypoparathyroidism, using a cell culture nutrient solution before gland transplantation.

Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the “gold standard” for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [13C315N1]-pantothenic acid, mammalian cells exclusively incorporated [13C315N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [13C315N1]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters

Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [(13)C(3)(15)N]-pantothenate (vitamin B(5)), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2-3 weeks.

Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [13C315N1]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [13C315N1]-pantothenate after three passages of the murine cells in culture. Charcoal−dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Electrostatic Charge Activates Inflammatory Vanilloid (VR1) Receptors

The pathophysiology of neurogenic inflammation culminates in the overt symptoms of tissue inflammation through a series of events which are initiated by the activation of vanilloid receptors (VR1). This study was designed to test the hypothesis that a sufficiently negative, electrostatic charge carried on a particulate matter (PM) particle, could acquire a cloud of protons sufficient to activate proton-sensitive VR1 receptors and acid-sensitive ionic channels (ASICs) pathways. For this, nanometer-sized, synthetic polystyrene micells (SPM) or those charged with chemical groups (e.g. diamino, carboxyl) were used. These chemical groups imparted either a net positive (i.e. diamino) or negative (i.e. carboxyl) charge on the SPM when suspended in a neutral ionic medium. The zeta potential, a measure of the SPM’s electronegativity, was taken in both cell culture nutrient medium and in ultraviolet light-distilled water (UV-DW). In both vehicles, the rank order of electronegativity (most to least negative) was carboxyl > polystyrene > diamino-SPM. Individual types of SPM were exposed to human, immortalized bronchial-tracheal epithelial cells (i.e. BEAS-2B) and endpoints of biological activation (i.e. membrane depolarization, increases in intracellular calcium (i.e. [Ca 2+] i ) levels, IL-6 release) were measured. Cells loaded with a fluorescent probe for membrane depolarization (3,3′-dihexyloxacarbocyanine iodide, DiOC-6-3) showed a positive reaction when exposed to carboxyl-SPM but not to diamino-SPM. BEAS-2B cells exposed to carboxyl-SPM responded with significant increases in [Ca 2+] i , and IL-6 release relative to uncharged SPM or diamino-SPM. This IL-6 release could be reduced by pretreatment with antagonists to the VR1 receptor (i.e. capsazepine) or to acid-sensitive ionc channels (i.e. amiloride). Although both diamino and carboxyl-SPM groups stimulated increases in IL-6 transcript, only the more electronegatively charged carboxyl-SPM stimulated mRNA–VR1 receptor. These data suggest that measurable inflammatory changes can be stimulated in human epithelial target cells by the electrostatic charge carried on an inert particle. Further, these changes appear to be mediated through acid-sensitive VR1 receptors and ASICs.

Serum from outdated human platelet concentrates: an alternative supplement for tissue (fibroblast) culture media.

Fetal calf serum (FCS) is used as a component of the media for cells grown in culture. However, the high cost of FCS has stimulated a search for a possible alternative. Approximately 10% of human platelet concentrates prepared for clinical transfusions are outdated. Sera prepared from platelet concentrates were compared with FCS and calf serum (CS) as a media supplement. A similar number of fibroblast (NSF-791) colonies grew in media supplemented with the three different sera. However, colonies grown in the presence of human platelet serum (HPS) were larger than those grown in media containing CS and FCS. Hence, serum prepared from outdated human platelet concentrates may be substituted for fetal calf serum as a cell culture nutrient, for fibroblasts.

In vitro cultivation of Eimeria acervulina (Coccidia)

Eimeria acervulina oocysts were sterilized with 50 ppm chlorine and viable sporozoites were released mechanically with the aid of trypsin and bile. These sporozoites, when suspended in the appropriate cell culture nutrient, were used as inoculum for chick embryo kidney, chick embryo fibroblast, mouse fibroblast, human amnion, and HeLa cell cultures. Infection of the host cell by sporozoites occurred in cultures of each cell type. Apparent cell infection occurred as early as one hour post inoculation, and forms recognized as trophozoites were observed at 18 to 24 hours. Further development failed to occur.