Browsing by Subject "Cell culture"

Now showing 1 - 6 of 6
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    Cell-culture Data Pipeline Python package (CDPpy) for processing and analyzing cell culture datasets
    (2024-08-05) Lu, Yen-An; Fukae, Yudai; Hu, Wei-Shou; Zhang, Qi; qizh@umn.edu; Zhang, Qi; University of Minnesota Decision Discovery and Optimization Lab; University of Minnesota Cellular Engineering Lab
    CDPpy (Cell-culture Data Pipeline Python package) is an open-source library designed for the analysis of fed-batch cell culture data from multiple experiments and cell lines. The package features the functions of a data processing pipeline and visualization toolbox. The processing pipeline reads raw data from Excel files following a fixed template, derives variables such as cumulative substrate consumption and various specific rates, and exports the processed dataset into an Excel file. The specific rates show changing cellular activities over time in culture, providing insights for process optimization. The visualization toolbox enables users to analyze process profiles across experimental runs and cell lines, aiding in future experimental design. In this repository, we include the source code for the package, an instruction for package setup, and a Jupyter notebook that provides step-by-step guidelines for data processing and visualization using an example dataset. The updated version will be announced in the GitHub repository: https://github.com/ddolab/CDPpy in the future.
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    Determining the Effect of Cell Culture Methods on the Polarization and Phenotype of Macrophages
    (2024) Lambrecht, Daniel; Chiu, Maggie; Provenzano, Paolo P.
    Macrophages play an important role in the regulation of cancer tumor microenvironments (TME). The specific role they play depends on their polarization, which is divided into two general phenotypes: M1, a pro-inflammatory phenotype, and M2, an anti-inflammatory phenotype known to support tumor growth. As the field of cancer research develops, there is an increased focus on understanding the role of the macrophage in the TME and how it can be manipulated to limit the growth of the tumor. Thus, it is important for researchers who are studying the macrophage’s role in the TME to know the phenotype of the macrophages that they are culturing in their research. The goal of this study was to observe the differentiation and polarization of macrophages during the standard cell culture protocol. We expected the macrophages to be fully differentiated after 7 days of culture with M-CSF and that cell passaging would result in a higher abundance of M1 polarized macrophages in culture. However, we found that macrophages are fully polarized after only 5 days of exposure to M-CSF and that passaging has no significant effect on macrophage polarization. This implies that macrophage differentiation protocols can be shortened with no loss in macrophage yield and that passaging is a suitable cell culture method for macrophages.
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    Fundamentals of a systems biology approach to In Vitro tissue growth
    (2013-05) Beck, Richard Joseph
    Tissue engineering needs a paradigm shift in order to generate clinically useful products. The field has yet to regularly produce implantable tissue-engineered products. The conventional manner in which input stimuli are applied without consideration of current cellular activity level is certainly suboptimal. The objective of this line of research is to produce a method for rationally choosing input stimuli that drive the cells toward optimal tissue growth. Transient phosphorylation of signaling proteins after a perturbation in stimuli contains biological information concerning downstream tissue growth. The overall project aims to build a statistical model predictive of tissue growth via information of the upstream phosphoproteome minutes after a change in stimuli. The validity of such a statistical model can be tested based on its utility to direct tissue growth: stimuli will be chosen on the basis of which corresponding phosphoproteome profile(s) is predicted to yield the best downstream tissue growth; this can be directly compared to conventional tissue engineering methods. This doctoral project focused on obtaining sample types and tailoring methods appropriate for a systems biology and statistical approach, especially in regard to the label-free quantification of phosphopeptide enrichments. Neonatal human dermal fibroblasts (nhDF) were expanded to near confluence, at which point basal medium for tissue production was applied. After two days, nhDF were perturbed with basal medium supplemented with 1 or 10 ng/mL TGF-β1. Cells were harvested at 10, 20, or 30 minutes for intracellular proteins. Resultant protein lysates were digested to peptides via trypsin and enriched for phosphopeptides via Iron Immobilized Metal Affinity Chromatography (IMAC). Phosphopeptide enrichments were analyzed by tandem mass spectrometry. A total of 1689 peptides were both identified with phosphorylation and quantified using distinct algorithms. Under strict statistical tests, 22 of these peptides were found to differ between treatments/time. Corresponding downstream collagen deposition was also found to differ between treatments. These results indicate that the type of quantitative data needed for the overall project can be acquired. The methods developed can be used in finding a statistical relationship between tissue growth and upstream phosphoproteome profiles.
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    A mechanistic-empirical model of central metabolism, signaling, and the reactor environment for bioprocesses
    (2020-10-07) O'Brien, Conor M; Hu, Wei-Shou; acre@umn.edu; Hu, Wei-Shou
    This model was built and optimized to reproduce the variability inherent to many industrial cell-culture processes. Classically, fed-batch Chinese Hamster Ovary (CHO) cell cultures will initially produce lactate in the early phase of culture before switching to lactate consumption. However, some processes may revert to lactate production in the late stage of culture, driving up osmolarity while reducing viable cell density, and ultimately lowering process performance. This phenomenon may occur in only some runs of a manufacturing processes and even may differ among runs with similar initial conditions and trajectories, leading to longstanding questions about the mechanisms driving this switch. By simulating cultures which were exposed to different amounts of stress before the production bioreactor we show that similar starting conditions in the bioreactor environment can lead to variability in metabolic shift. We provide this model as a tool to demonstrate this metabolic variability and provide a platform for hypothesis testing, in silico bioprocess optimization, and simulation of reactor scale-up and scale-down.
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    Morphological characterization of medium spiny neuron development in vitro
    (2012-05) Penrod-Martin, Rachel Dobrof
    Medium Spiny Neurons (MSNs) are the primary cell type of the striatum, a structure critically involved in motivation, memory, and movement. MSN structure and function are altered in Parkinson’s and Huntington’s disease as well as following repeated exposure to drugs of abuse. One site on the MSN that changes in all of these diseases is the dendritic spine, a sub-micron protrusion responsible for receiving excitatory (glutamatergic) signals. Dendritic spines undergo structural and functional changes during development, normal aging, and disease. To date, no studies have specifically characterized the developmental changes undergone by MSN dendritic spines and investigation into the molecules that regulate MSN dendritic spines has been limited to in vivo manipulations in adults or characterization of global knockout of specific molecules. Research into MSN dendritic spine development and plasticity mechanisms were likely limited by the absence of an in vitro primary neuronal culture system that produced MSNs with mature in vivo-like characteristics. The experiments detailed in this thesis describe the development and characterization of such a system as well as a morphological analysis of MSN development in culture. MSNs cultured using this technique develop gross morphological characteristics similar to in vivo cells. They also develop high densities of dendritic spines with a high variety of mature morphologies and have synaptic and intrinsic physiological characteristics like MSNs in vivo. This work represents a significant contribution to the field, supporting future research into the molecules that regulate MSN development and plasticity in both normal and disease models.
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    Pharmacogenomic modeling of bortezomib resistance in B cell malignancies
    (2013-04) Stessman, Holly Annette-Feser
    Proteasome inhibitors are a class of drugs that have been largely successful in the treatment of cancer patients, particularly those with the plasma cell malignancy, multiple myeloma. The most successful of these drugs, bortezomib (Bz), has paved the way for the development of next-generation proteasome inhibitors. Although Bz has significantly contributed to improved outcomes in myeloma patients, acquired resistance to Bz is imminent. Furthermore, a portion of patients never initially respond to the drug. Therefore, the goal of these studies was to further characterize Bz resistance with the aim to better predict secondary therapies that may be used successfully with Bz to recapture drug sensitivity.In the first study, we describe the creation of an in vitro malignant mouse plasma cell system from which we create isogenic pairs of Bz-sensitive and -resistant cell lines. We further characterize the transcriptional responses of these cell line pairs to identify both conserved and unique expression signatures. Using the expression signatures that are unique to each pair of cell lines, we identify secondary therapies that may be useful for treatment of the Bz-refractory cell line using an in silico database called Connectivity Map (CMAP). This analysis predicted a unique response to histone deacetylase inhibitors, a class of drugs that are currently being tested for efficacy in myeloma, in only one mouse cell line pair. Indeed, we find that the predicted Bz-resistant cell line has increased sensitivity to this class of drugs (including the drug panobinostat). When these cells were transferred back into syngeneic recipient mice, panobinostat treatment could successfully extend the life of Bz-resistant animals suggesting that the Bz-resistant phenotype may select also for increased sensitivity to other drugs that may be identified through in silico approaches. In the second study, we follow up these observations by investigating other CMAP prediction patterns, such as those that are conserved across all cell line pairs. A second prediction of one class of these CMAP-predicted drugs using high-throughput drug screening of the cell lines revealed that a combination of these approaches may be highly successful for accurate prediction of secondary therapies. Based on these predictions, we further investigate the efficacy of topoisomerase inhibitors in combination with Bz for the treatment of Bz-resistant cell lines.In the third study, we provide further immunophenotypic characterization of the Bz-sensitive and -resistant mouse cell lines revealing not only cell surface markers that are associated with "acquired" and "innate" Bz resistance but perhaps a mechanism of resistance. Although Bz-sensitive mouse cells display a classic myeloma phenotype, homing to the bone marrow in vivo and expressing classic plasma cell markers, Bz-resistant mouse cells present as extramedullary disease and express a more B cell-like immunophenotype. We identify that differences in migration may be linked to the differential expression of the bone marrow homing protein, CXCR4. Lower expression of this gene in a Bz human clinical trial was also associated with inferior survival. Immunophenotypic characterization of these cell populations further revealed that forced differentiation of the Bz-resistant population could restore Bz-sensitivity.The final study investigates the acquisition of Bz-resistance in a B cell malignancy, Burkitt lymphoma, that is currently undergoing Bz clinical trials. In this particular malignancy, a DNA mutator, AID, is known to be expressed that may contribute to other types of drug resistance. Here, we identify that this is unlikely a mechanism for developing resistance to Bz. Furthermore, we provide evidence that AID activity is reduced in Bz-resistant clones and, in fact, that high AID expression may be selectively eliminated during Bz selection.