Method For Isolation Of Cells Research Articles

Blood Collection Workflow for Interrogation of Translational Samples with Single Cell Technologies

Blood is a valuable and easily accessible sample type for clinical researchers that provides a snapshot of the organ systems at the time of sample collection. Readouts obtained from blood help inform clinical treatments, investigate disease mechanisms, evaluate drug response, and monitor outcomes. However, the impact of existing blood collection workflows on data output from new and high resolution technologies, like single cell RNA sequencing (scRNA-seq) remains a challenge. Recent scRNA-seq studies have shown that changes in gene expression can occur in blood cells in as little as a few hours post collection1. While bulk measurements rely on sample fixation to preserve the sample during transport, this approach does not preserve intact cells and hence, is not compatible with single cell technologies. With the recent availability of fixation compatible scRNA-seq assays comes the need to preserve blood while retaining single cell information. In this study, we evaluated various PBMC isolation and blood preservation methods which preserve single cell information while easing sample logistical constraints. Our initial experiments compared various PBMC isolation methods. scRNA seq analysis of these samples demonstrated that all isolation methods captured single cell gene expression with some variability in cell types along with user-variability. Changes in cell type proportions were observed in as little as 4 hrs post blood draw, emphasizing the need for an effective blood preservation method to retain single cells during transport and blood cell isolation. Next, we compared different methods of blood preservation including cryopreservation and fixation. Healthy and diseased whole blood samples were collected, preserved, stored followed by isolation of blood samples at different time points. scRNA-seq analysis of the samples demonstrated that blood can be preserved, transported, and stored (weeks to months) before blood cell isolation. Blood cells isolated from these cells can then be stored long term for scRNA-seq analysis. This blood collection and preparation workflow also eases blood transportation logistical constraints, allowing for batch shipping of samples from distributed collection sites. Coupled with automatable cell isolation methods and multiplexing single cell sequencing solutions, these workflows can be adapted for large scale translational research studies.

Ultracentrifugation method for exosome isolation of HEK293 cells as a candidate model in-vitro therapy of breast cancer

Abstract. Amelida RN, Mariya S, Zaelani BFD, Yuliana, Dayana H, Darusman HS. 2023. Ultracentrifugation method for exosome isolation of HEK293 cells as a candidate model in-vitro therapy of breast cancer. Biodiversitas 24: 5074-5079. Exosomes are secreted by all cells and found in all body fluids, providing the biological system. They have great potential in cancer immunotherapy and have different roles and targets. The biological function of an exosome relies on its bioactive activities, supported by lipids, proteins, and nucleic acids. Exosomes can be isolated by the ultracentrifugation method, widely used to produce exosomes from various sources and intended to pellet containing heterogeneous particles and the exosomes. In this study, we isolated exosomes derived from HEK293 cells using the ultracentrifugation method and applied directly in vitro to MCF-7 cells. The exosomes were added to the MCF-7 cells at various concentrations, 3,5 µg/mL, 7 µg/mL, and 14 µg/mL, evaluated using P53 and PCNA markers. The study showed that the exosomes had been successfully isolated; the concentration obtained was 24,95 µg/mL and expressed exosome markers CD9 and CD81. The MCF-7 cells were treated by exosome and demonstrated epithelial-like morphology. The cell population survived, but the confluency decreased at higher concentrations, as did viability. The marker of P53 and PCNA expression resulted from the effect of exosome treatment. The results indicate that the exosome can potentially inhibit the growth of breast cancer cells in vitro.

Methods for cell isolation and analysis of the highly regenerative tunicate Polycarpa mytiligera.

Background: Polycarpa mytiligera is the only molecularly characterized solitary ascidian capable of regenerating all organs and tissue types. The cellular basis for regeneration in P. mytiligera is largely unknown, and methods for isolating live cells from this species for functional analyses are unavailable. Results: Here, we developed a method for isolating live cells from P. mytiligera, overcoming major experimental challenges, including the dissociation of its thick body wall and native cellular autofluorescence. We demonstrated the applicability of our approach for tissue dissociation and cell analysis using three flow cytometry platforms, and by using broadly used non-species-specific cell labeling reagents. In addition to live cell isolation, proof-of-concept experiments showed that this approach was compatible with gene expression analysis of RNA extracted from the isolated cells, and with ex vivo analysis of phagocytosis. Conclusion: We presented efficient methods for cell purification from a highly regenerative ascidian, which could be transferable to diversity of non-model marine organisms. The ability to purify live cells will promote future studies of cell function in P. mytiligera regeneration.

Urine-derived stem cell therapy for diabetes mellitus and its complications: progress and challenges.

Diabetes mellitus (DM) is a chronic and relentlessly progressive metabolic disease characterized by a relative or absolute deficiency of insulin in the body, leading to increased production of advanced glycosylation end products that further enhance oxidative and nitrosative stresses, often leading to multiple macrovascular (cardiovascular disease) and microvascular (e.g., diabetic nephropathy, diabetic retinopathy, and neuropathy) complications, representing the ninth leading cause of death worldwide. Existing medical treatments do not provide a complete cure for DM; thus, stem cell transplantation therapy has become the focus of research on DM and its complications. Urine-derived stem cells (USCs), which are isolated from fresh urine and have biological properties similar to those of mesenchymal stem cells (MSCs), were demonstrated to exert antiapoptotic, antifibrotic, anti-inflammatory, and proangiogenic effects through direct differentiation or paracrine mechanisms and potentially treat patients with DM. USCs also have the advantages of simple noninvasive sample collection procedures, minimal ethical issues, low cost, and easy cell isolation methods and thus have received more attention in regenerative therapies in recent years. This review outlines the biological properties of USCs and the research progress and current limitations of their role in DM and related complications. In summary, USCs have shown good versatility in treating hyperglycemia-impaired target organs in preclinical models, and many challenges remain in translating USC therapies to the clinic.

Isolation and Culture of Human Umbilical Vein Endothelial Cells (HUVECs).

This protocol describes a simple and an economical method for isolation of endothelial cells from human umbilical vein. Umbilical cord is easily available postpartum following informed consent, and the method for its collection is noninvasive with few ethical concerns. Thus, umbilical vein is an ideal source for isolation of endothelial cells of human origin. Endothelial cells are isolated from umbilical vein by collagenase digestion. This is followed by their culture on extracellular matrix (ECM) protein coated tissue culture flasks in presence of endothelial cell growth medium. In the last few decades, human umbilical vein endothelial cells (HUVECs) have come to be regarded as a standard model system by vascular biologists to understand general principles of endothelial cell biology and dysfunction. Here, we describe isolation, culture, as well as validation of HUVECs.

An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeability Studies.

The blood-retinal barrier (BRB) present in the posterior chamber of the eye plays a major role in maintaining the proper function and integrity of the retina. Retinal pigment epithelium and choriocapillaris form the outer blood retinal barrier (oBRB), and breakdown of this barrier leads to vision-threatening diseases like macular edema, macular degeneration, and diabetic retinopathy. A simplified cell culture model of oBRB will be of great importance in elucidating the molecular mechanism of the disease progression. This chapter describes methods for primary cell isolation from donor eyes to culture human retinal pigment epithelial cells (hRPE) and choroidal endothelial cells (hCEC) and the protocol for construction of a simplified in vitro model of oBRB on fibronectin-coated Transwell inserts. Further, we explained the permeability study using FITC-dextran conjugated tracers for validating the bilayer model. The permeability experiments ensured that the system could easily be manipulated to recapitulate the pathological condition in vitro. Thus, it would be an optimal system for studying the disease mechanisms related to retinal and choroidal pathologies, for screening small molecules, and for performing drug permeability kinetics. Moreover, fundamental understanding of paracellular and transcellular trafficking of cargo in hRPE and hCEC could also be studied using this model.

Effects of various xenogenic mesenchymal stem cell secretome fractions on the regenerative capacity of the liver in vitro

Liver diseases cause many deaths worldwide and wreak havoc on the economy. The main hepatoprotectors are plant substances and peptides. Regenerative medicine based on mesenchymal stem cells (MSCs) can offer a new set of biologically active substances for liver regeneration, which are part of their secretome. The work applied the methods of cell isolation and cultivation, ultrafiltration for fractionation of secretome components, and organotypic culture model. The study’s results indicate an increase in the regenerative capacity of the liver under the impact of the components of the MSC secretome; the fraction below 10 kDa shows the most significant activity. Keywords: organotypic liver culture; xenogenic stem cells; fractions of secretome; absorption spectrum, ultrafiltration

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

A Novel Method for Human Adipose-Derived Stem Cell Isolation and Cryopreservation.

Adipose-derived stem cells (ADSCs) are isolated from abundant adipose tissue and have the capacity to differentiate into multiple cell lineages. ADSCs have raised big interest in therapeutic applications in regenerative medicine and demonstrated to fulfill the criteria for a successful cell therapy. There are several methods for isolation of ADSCs from adipose tissue and cryopreservation of ADSCs. Here, novel methods for the isolation and cryopreservation of ADSCs are presented and focused. Microscopic pieces of adipose tissue were placed on transwell inserts, and the ADSCs were induced to migrate to the lower wells for 1 week. We compared the properties of our ADSCs with those isolated by enzymatic digestion and enzyme-free method of culture plate, and our ADSCs were found to be more stable and healthier. In addition, we proposed a novel cryoprotectant solution (FNCP) containing pectin and L-alanine, which was compared with standard cryoprotectant solution. Overall, our methods proved more useful for ADSCs isolation than other methods and did not require consideration of "minimal manipulation" by the U.S. Food and Drug Administration (FDA). Furthermore, our FNCP did not contain dimethyl sulfoxide and fetal bovine serum, therefore stable storage is possible in xeno-free and animal-free cryopreservation solutions.

Murine Dermal Lymphatic Endothelial Cell Isolation.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes. Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Ultrasound cavitation: a reliable non-enzymatic method for adipose-derived mesenchymal stem cell (ADSC) isolation

BackgroundAdipose tissue is known to serve as an abundant and readily accessible source of adipose-derived stem cells (ADSCs) as an alternative to bone marrow. Collagenase is one of the most widely used methods for the isolation of ADSCs from adipose tissue, but it takes a long time, and there are also debates about safety. We propose an ultrasonic cavitation-treated method that can significantly reduce time and avoid the problem of using xenogeneic enzymes in ADSCs isolation.MethodsADSCs were isolated from adipose tissue using the enzyme treatment method and the ultrasonic cavitation treatment method. Cell proliferation was measured using cell viability assay. The expression levels of the surface markers of ADSCs were estimated by real-time PCR. After, ADSCs were cultured in chondrogenic, osteogenic, or adipogenic differentiation medium; the differentiation potential of ADCSs was analyzed by Alcian blue, Alizarin Red S, Oil Red O, and real-time PCR.ResultsThe cells treated with collagenase and ultrasound had similar cell yields and proliferation after isolation. The difference in the expression of surface markers of ADSCs was not statistically significant. ADSCs showed differentiation potential into adipocytes, osteocytes, and chondrocytes, and there was no difference between the enzyme treatment method and the ultrasonic cavitation treatment method. The yield of the ADSC increased in time- and intensity dependently.ConclusionsUltrasound certainly serves as a promising method in advancing ADSC isolation technology.

An optimized force-triggered density gradient sedimentation method for isolation of pelage follicle dermal papilla cells from neonatal mouse skin

BackgroundThe dermal papilla cells are a specialized population of mesenchymal cells located at the base of the hair follicle (HF), which possess the capacity to regulate HF morphogenesis and regeneration. However, lack of cell-type specific surface markers restricts the isolation of DP cells and application for tissue engineering purposes.MethodsWe describe a novel force-triggered density gradient sedimentation (FDGS) method to efficiently obtain purified follicular DP-spheres cells from neonatal mouse back skin, utilizing only centrifugation and optimized density gradients.ResultsExpression of characteristic DP cell markers, alkaline phosphatase, β-catenin, versican, and neural cell adhesion molecules, were confirmed by immunofluorescence. Further, the patch assays demonstrated that DP cells maintained their hair regenerative capacity in vivo. Compared with current methods, including microdissection and fluorescence-activated cell sorting, the FDGS technique is simpler and more efficient for isolating DP cells from neonatal mouse skin.ConclusionsThe FDGS method will improve the research potential of neonatal mouse pelage-derived DP cells for tissue engineering purposes.

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells.

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

The control of breathing in fishes – historical perspectives and the path ahead

The study of breathing in fishes has featured prominently in Journal of Experimental Biology (JEB), particularly during the latter half of the past century. Indeed, many of the seminal discoveries in this important sub-field of comparative respiratory physiology were reported first in JEB. The period spanning 1960-1990 (the 'golden age of comparative respiratory physiology') witnessed intense innovation in the development of methods to study the control of breathing. Many of the guiding principles of piscine ventilatory control originated during this period, including our understanding of the dominance of O2 as the driver of ventilation in fish. However, a critical issue - the identity of the peripheral O2 chemoreceptors - remained unanswered until methods for cell isolation, culture and patch-clamp recording established that gill neuroepithelial cells (NECs) respond to hypoxia in vitro. Yet, the role of the NECs and other putative peripheral or central chemoreceptors in the control of ventilation in vivo remains poorly understood. Further progress will be driven by the implementation of genetic tools, most of which can be used in zebrafish (Danio rerio). These tools include CRISPR/Cas9 for selective gene knockout, and Tol2 systems for transgenesis, the latter of which enables optogenetic stimulation of cellular pathways, cellular ablation and in vivo cell-specific biosensing. Using these methods, the next period of discovery will see the identification of the peripheral sensory pathways that initiate ventilatory responses, and will elucidate the nature of their integration within the central nervous system and their link to the efferent motor neurons that control breathing.

PRIMARY CULTURE AND IDENTIFICATION OF MOUSE BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Brain microvascular endothelial cells are the basic components of the blood-brain barrier. Many neurological diseases are related to the loss of blood brain barrier (BBB) function. Isolating and culturing primary BMVEC is an important means to study the function and regulation of BBB in vitro. To establish a method for isolation and culture of primary mouse brain microvascular endothelial cells (BMECs). The 7-10-days mice were sacrificed by neck removal, the cranial cavity was opened, the brain was aseptically removed and the cerebral cortex was retained. To extract brain microvascular segments, the brain underwent three D-Hank solution rinses. It was then homogenized, twice digested by enzyme, and centrifuged using a Bovine Serum Albumin (BSA) gradient. For primary culture, the brain microvascular segments were injected into gelatin-coated culture plates using DMED complete media. When the cell density reaches 90 %, the media is removed, and the cells are then given two PBS washes. A new medium was introduced after adding 1ml of the trypsin-EDTA solution to digest for 2–5 minutes for passage. Cell culture plates were rinsed and pre-cooled 95 % ethanol was added for 20 minutes after passaged cells had grown to 80–90 % of their original size. After a third wash, 1 ml of mouse factor VIII antibody was added to the culture wells, where it was left for 4 hours at 37 °C. FITC-labeled rabbit anti-mouse antibody in a volume of 1 mL was added. Under a confocal laser microscope, the plates were examined and taken pictures of. The outcome demonstrates positive expression of the marker factor VIII associated antigen. To create a cell suspension, monolayer-growing microvascular endothelial cells were chosen. 100 L of cell suspension was used to inoculate each well in 96-well plates, and the cells were then grown. On days 1, 2, 3, 4, 5, 6, and 7, the original medium was removed, and then 180 μL of DMEM and 20 μL of MTT were simultaneously added to each well for a 4-hour culture. After adding dimethyl sulfoxide (DMSO), each well's absorbance value (A value) was measured at 492 nm. The outcome demonstrates that the growth peak is attained between 6 and 8 days. This method can successfully isolate and culture primary BMECs, which lay a foundation for the study of BMEC in vitro.

Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Design and simulation of a novel integrated microfluidic chip for cell isolation and culture.

The lab on a chip is utilized as a background and a substrate for creating a proper flow for cellular processes in medicine. In this study, the concepts of cell isolation and cell transfer methods have been discussed. After that, the device of separation and transfer systems has been designed, simulated, and verified by placing the frequency of particle separation and droplet formation, which is tried to introduce a new device that can be used in cellular studies. The optimal operation conditions for the problem have also been investigated. High separation efficiency (99%) could be achieved when the velocity of the sample inlet in the microchannel separator is 180μm/s. Also, a microfluidic device for droplet generation has been designed to transfer the isolated cells to the culture medium. For this purpose, the frequency of droplet production must be synchronized with particle ejection frequency and equals 9.09Hz.

Phenotyping senescent mesenchymal stromal cells using AI image translation.

Mesenchymal stromal cells (MSCs) offer promising potential in biomedical research, clinical therapeutics, and immunomodulatory therapies due to their ease of isolation and multipotent, immunoprivileged, and immunosuppersive properties. Extensive efforts have focused on optimizing the cell isolation and culture methods to generate scalable, therapeutically-relevant MSCs for clinical applications. However, MSC-based therapies are often hindered by cell heterogeneity and inconsistency of therapeutic function caused, in part, by MSC senescence. As such, noninvasive and molecular-based MSC characterizations play an essential role in assuring the consistency of MSC functions. Here, we demonstrated that AI image translation algorithms can effectively predict immunofluorescence images of MSC senescence markers from phase contrast images. We showed that the expression level of senescence markers including senescence-associated beta-galactosidase (SABG), p16, p21, and p38 are accurately predicted by deep-learning models for Doxorubicin-induced MSC senescence, irradiation-induced MSC senescence, and replicative MSC senescence. Our AI model distinguished the non-senescent and senescent MSC populations and simultaneously captured the cell-to-cell variability within a population. Our microscopy-based phenotyping platform can be integrated with cell culture routines making it an easily accessible tool for MSC engineering and manufacturing.

Comparison of methods to isolate peripheral blood mononuclear cells from cattle blood

Peripheral blood mononuclear cells (PBMCs) are critical for assessment of host immune responses to infectious disease. The isolation of PBMCs from whole blood is a laborious process involving density gradients and multiple centrifugation steps. In the present study we compared a more traditional method of PBMC isolation used in our laboratory to two novel methods of cell isolation for efficiency, cell viability, and enumeration of cell subsets. Our laboratory method uses Histopaque-1077 density gradient in standard conical tubes and this was compared with isolation of cells using SepMate™ tubes, a novel conical tube containing an insert to separate the density gradient. Multiple experiments were performed to optimize the SepMate™ tubes for use with cattle blood. A final experiment was conducted to compare traditional methodology, the optimized SepMate™ method with a more novel method using cell preparation tubes (CPT-10 vacutainers containing density gradient). Results demonstrated that optimization of the SepMate™ tube methodology was necessary, including dilution of blood and addition of centrifugation steps to reduce platelet contamination. The CPT-10 tubes worked well but cell recovery was lower compared to other methods. Both of the newer methods were comparable to a modified version of our traditional laboratory method of PBMC isolation in terms of numbers of recovered viable cells and the frequency of immune cell subsets. Additionally, efficiency was improved, particularly with the SepMate™ tube method, resulting in reduced time in the laboratory as well as reduced usage of plasticware.

A Device for Isolation of Selected Single Adherent Cells.

In recent years, extensive research has been focused on the field of single cell analysis. The isolation of single cells is the first step in this type of research. However, the techniques used for direct isolation and acquisition of single adherent cells are limited. Here, we present a method of obtaining selected single adherent cells using a separation device. Compared with other single cell isolation methods, this method has the advantages of simple operation, low cost, minimal cell damage, and preservation of cell morphology. Our methodology is, therefore, suitable for the collection of selected single adherent cells.