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Human Tissue and Biofluids Procurement

FIDELIS RESEARCH recognizes the increasing importance of biospecimens, which are becoming essential tools for the translation of our client’s research into new products.

 

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Isolation of Peripheral Blood Mononuclear Cells (PMBCs)

Mononuclear cells refer to blood cells that have a single, round nucleus. When isolated from circulating blood, they are called peripheral blood mononuclear cells (PBMC), but other sources exist, such as the umbilical cord, spleen, and bone marrow. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nuclei, and granulocytes (neutrophils, basophils, and eosinophils) have multi-lobed nuclei. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, and only a small percentage of dendritic cells. These cells are critical components of the immune system which are involved in both humoral and cell-mediated immunity.

The established method for separating mononuclear cells from blood is through density gradient centrifugation using the polysaccharide Ficoll. Upon spinning, the cells are collected in a defined layer which comprise about 1% of the total sample volume. From this fraction, more specific cell types can be further isolated by different purification methods that basically target specific cell surface proteins (e.g. CD4 for T helper cells, CD8 for cytotoxic T cells, CD19 for B cells, etc.).

Mononuclear cells have been essential element and a powerful tool in the research areas of immunology, infectious disease, hematological malignancies, vaccine development, transplant therapy, personalised medicine, and toxicology. Through efficient and successful processing and analysis of PBMCs, researchers and clinicians can test immune responses, gain a deeper understanding of the immune system, and apply their findings to treatments and cures for human diseases.

Peripheral blood is obtained from fully consented and approved medical centers following strict ethical standards

FIDELIS RESEARCH offers diseased PBMCs from a wide variety of human diseases.

Isolation of Peripheral Blood Plasma

Plasma is the liquid part of the blood that carries cells and proteins throughout the body. It makes up about 55% of the body's total blood volume. It is mostly water (up to 95% by volume), and contains important dissolved proteins (6–8%) (e.g., serum albumins, globulins, and fibrinogen), glucose, clotting factors, electrolytes (Na+, Ca2+, Mg2+, HCO3, Cl, etc.), hormones, carbon dioxide, and oxygen.

Blood plasma is the most easily accessible source for biomarker discovery and the most preferred diagnostic material for disease proteomic and genetic studies due to its non-invasive nature. It is a heterogeneous collection of proteins secreted or leaked from all types of tissues revealing the cellular state due to spatio-temporal differences in protein expression. Thus, being a direct reflection of the patho-physiological condition of a patient, it is considered to be a diagnostic goldmine for biomarkers research.

The proteins constituting the plasma proteome can be categorized into three different classes. The first contains abundant (~95%) proteins with a functional role in blood. The second class are tissue leakage proteins without a dedicated function in the circulation. The third class are signaling molecules like small protein hormones (for instance, insulin) and cytokines, which typically have very low abundances at steady state and are upregulated when needed. However, it is this low abundance fraction of proteome that contains tissue leakage proteins and proteins derived from pathological sources containing information on the onset and progression of a disease.

Additionally, plasma may also contain another type of molecules such as extracellular RNAs (exRNAs), circulating cell-free DNA (cfDNA) released by diseased cells, allowing researchers the ability to identify biomarkers that aid in the development of cost-effective screens for the early detection and diagnosis of diseases. exRNA has emerged as an important source of biological information that represents the dynamic processes that occur intra- and intercellularly, in real time. exRNAs are released in a variety of subpopulations, with huge application as biomarkers for earlier cancer diagnosis, tumor progression monitor, and prediction of therapy response. Similarly, an increasing number of studies have demonstrated the use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. cfDNA is becoming an importance source for tumor mutation detection.

Another interesting component of the plasma, with emerging role in diagnosis, prognosis, and therapies of patients with cancer are the so-called exosomes. Exosomes represent a heterogenous population of vesicles produced by all cells, carry diverse molecular and genetic cargos and circulate freely through body fluids. Cancer patients’ plasma contains exosomes produced by malignant and normal cells. Exosomes are currently emerging as promising noninvasive correlates of tumor progression and as markers of immune evasion of cancer.

Plasma is separated from peripheral blood by centrifugation.

Blood plasma remains the predominant biological matrix to assess health and disease in clinical settings. Many studies have emphasized the importance of plasma as a treasure-trove for biomarker discovery.

Sample quality is critical for biomarker detection in oncology. Fidelis Research Теam guarantees highly professional sample collection and further processing.

Flow Cytometry

Flow cytometry (FC) is an immunophenotyping technique in which suspensions of living cells are stained with specific, fluorescently labeled antibodies and then analyzed with a flow cytometer.

Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, measuring of cell features, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analyzing cell size and volume. The power of flow cytometry is that it allows simultaneous multi-parameter analysis of single cells. This technique allows researchers to get highly specific information about individual cells. The most critical requirement for efficient and effective flow cytometry analysis is that the sample be a single-cell suspension. This helps ensure that every cell is analyzed independently.

FIDELIS RESEARCH provides flow cytometry services from experimental design to specimen processing, acquisition, and data analysis. We can customize the flow cytometry assays to meet our client’s needs

We offer you a flow cytometry service solution for a wide variety of applications:

  • Immunophenotyping
  • Antigen Specific Responses
  • Intracellular Cytokine Analysis
  • Proliferation Analysis
  • Cell Cycle Analysis
  • Signal Transduction
  • Cell health status—from viability to late-stage apoptosis or programmed cell death
  • Identification and characterization of distinct subsets of cells within a heterogeneous sample

We are ready to provide solutions to your flow cytometry needs in your next stage of research.

Positive Immunomagnetic Separation of CD34+ Cells

Using positive immunomagnetic separation techniques, FIDELIS RESEARCH is successfully isolating CD34+ cells, which are characterized by high purity and yield. The positively isolated cells are bead and antibody-free, phenotypically unaltered and suitable for any downstream applications including flow cytometry, functional studies, and cell culturing.

CD34 is a glycosylated transmembrane protein and represents a well-known marker for primitive blood- and bone marrow-derived progenitor cells. CD34+ stem cells are multipotent and can give rise to all cell types in blood. Additionally, CD34+ cells are responsible for all lymphohematopoietic lineages even though they comprise only a small percentage of the cell population.

The isolation of CD34+ cells from human umbilical cord blood (CB) is an important step in hematopoietic stem and progenitor cell (HSPC) research.

Besides the stem cell research, there is an extensive interest to the CD34+ cells in regard to exploring the pathogenesis processes of acute myeloid leukemia (AML). AML is generally regarded as a stem cell disease. It originates from a class of leukemic stem cells that are capable of self-renewal. The leukemia stem cells (LSCs) in AML play an important role in the development, relapse, and progression of leukemia, and in the development of chemotherapeutic drug resistance. It is well known that the stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with AML.

Fidelis Research is effectively partnering with leading biotech companies which are conducting research and studies with CD34+ cells.

Isolation of Cells (Single Cells and Spheroids) from Malignant Pleural Effusions and Ascites

Malignant pleural effusion (MPE) and ascites (MA), common clinical manifestations in advanced cancer patients, are associated with a uniformly fatal prognosis and a life expectancy of only 3 to 12 months. For that reason, the development of an effective targeted therapy represents a pressing unmet need.

MA and MPE constitute a unique tumor microenvironment providing a physical structure for the accumulation of cellular and acellular components. MA and MPE are initiated and maintained by physical and biological factors resulting from underlying disease and forms an ecosystem that contributes to disease progression. It has been demonstrated that the cellular contents and the molecular signatures of MA and MPE change continuously during the course of a disease. Over the past years, increasing attention has been given to the characterization of components of MA and MPE and their role in the progression of several types of cancer.

The importance of the tumor microenvironment in cancer progression has been increasingly recognized and it plays an essential role in mediating and sustaining the hallmarks of cancer. Expanding our knowledge of both the cellular (tumor cells and stromal cells) and acellular (soluble factors) component of MA and MPE is extremely important. All of these components work in tight coordination to create tumor-friendly microenvironments, which fosters the acquisition of hallmarks. It is scientists’ task to harness strengths and implement new arsenal in the fight against cancer.

 

FIDELIS RESEARCH can provide prospective collection of malignant pleural effusion and ascites fluids from patients with various cancers (lung, breast, ovarian, colorectal cancers, and so on) and the subsequent isolation of viable cryopreserved cells from them. Additionally, FIDELIS RESEARCH can provide further separation of the isolated cells into two subpopulations – single cells and aggregated tumor cells, or the so-called spheroids.

 

Biofluids are obtained fully consented from approved medical centers following strict ethical standards.

Malignant pleural effusion and ascites are collected and transported to Fidelis Research laboratories within two to four hours.

RNA & DNA Purification

The extraction of DNA&RNA is a starting point and an essential requirement for all downstream genomic and transcriptomic analyses. FIDELIS RESEARCH provides DNA and RNA purification services from a wide variety of starting material to meet the demands of any project. We have experience with genomic DNA, total RNA, miRNA, cell free circulating DNA/RNA isolation from nearly all biomaterials including whole blood, bone marrow, tissues, buffy coats, PMBCs, nuclear pellets, serum, saliva, mouthwash, buccal swabs, FFPE blocks etc. Our nucleic acid purification protocols are optimized to provide maximum yield, purity, and integrity from virtually any sample type. The extracted DNA and RNA fulfills the highest demands of quality. Our procedure includes Quantification and Quality assessment of the extracted material.

Different DNA/RNA types that FIDELIS RESEARCH can extracts are:

I. Isolation of miRNA

Purification of cell-free total RNA, including miRNA, from small volumes of human plasma and serum.

The extracellular miRNAs in serum and plasma are relatively stable molecules and in the recent years have demonstrated huge potential in their application as noninvasive biomarkers for a variety of diseases, including cancer.

The purified miRNA could be for use in a variety of downstream applications, including:

  • Biomarker discovery
  • Northern blot analysis
  • Quantitative, real-time RT-PCR
  • Microarray analysis

 a. miRNA from tissue and cells

Effective purification of miRNA and total RNA from cells and tissues. The purification protocol allows efficient enrichment of miRNA and RNAs with length smaller than 200 nucleotides. The obtained RNA is characterized with high-purity RNA suitable for all downstream applications

b. miRNA from exosomes

For purification of total RNA, including miRNA, from exosomes and other extracellular vesicles (EVs) human plasma and serum.

c. miRNA from FFPE

Purification of miRNA and total RNA that includes RNA from approximately 18 nucleotides upwards from formalin-fixed, paraffin-embedded (FFPE) tissue sections. The purified RNA can be further used in PCR, qPRC, real-time RT-PCR, microArray experiments.

 II. Isolation of ccfDNA

Isolation of free-circulating DNA from human plasma or serum.

Cell free-circulating DNA, such as tumor-specific extracellular DNA fragments in the blood or fetal DNA in maternal blood, is present in serum or plasma usually as short fragments of <1000 bp. An increasing number of studies demonstrate the potential use of circulating cell-free DNA as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring.

 III. Isolation of ccfDNA and ccfRNA

Co-purification of cell-free DNA and RNA (including miRNA), including vesicular and non-vesicular nucleic acids. The protocol allows for small elution volumes (of ~ 15-20 µl). Preparation of samples suitable for liquid biopsy when parallel analysis of DNA and RNA mutations is required or for samples with low mutant allele frequencies.

 IV. Isolation of genomic DNA

a. from body fluids (blood, plasma etc.)

Purification of genomic DNA from blood and related body fluids.

Sample sources may include:

  • Fresh and frozen whole blood or buffy coat
  • Plasma or serum
  • Bone marrow
  • Lymphocytes
  • Platelets
  • Body fluids    

b. from blood, tissue and cell cultures

Isolation of high-molecular-weight DNA from blood, tissue and cultured cells

DNA purified from Blood & Cell Culture is well suited for use in the different applications:

  • Sanger and next generation sequencing
  • RFLP analysis
  • Analysis of gene targeting
  • DNA fingerprinting studies
  • PCR amplification

c. from FFPE

Purification of genomic DNA from formalin-fixed paraffin-embedded tissues, guaranteeing consistent, high yields and complete removal of contaminants and inhibitors. The kit purifies DNA that can be used in a wide range of downstream applications: PCR,  single nucleotide polymorphism (SNP) and short-tandem repeat (STR) genotyping and pharmacogenomic research.

V. Isolation of total RNA

a. from cells / tissue 

Purification of total RNA from cells and tissues, delivering high-quality total RNA ready-to-use for high performance in any further downstream application. The purification protocol guarantees consistent RNA yields from small amounts of starting material.

b. from blood 

Purification of cellular RNA from fresh whole blood; consistent, high yields and removal of contaminants and inhibitors. The purified RNA can be further used for PCR, real-time PCR, microarray analysis.

c. from FFPE

For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections, applying novel method to overcome formalin crosslinking. The purification protocol guarantees efficient release of RNA without compromising integrity.

The used commercial kit isolates all RNA molecules longer than 70 nucleotides from FFPE samples, providing usable RNA fragments for numerous downstream applications, including RT-PCR. However, RNA purified from FFPE samples is heavily fragmented and should not be used in downstream applications that require full-length RNA. Some applications may require modifications to allow the use of fragmented RNA (e.g., designing small amplicons for RT-PCR).

Dissociation of Solid Tumor Tissues

For decades, immortal cancer cell lines have been extensively used as a robust biological model to investigate cancer biology and explore the potential efficacy of anticancer drugs. However, nowadays more and more studies support thе suggestion that these cell lines poorly represent the heterogeneity, diversity, and drug-resistant tumors occurring in patients. Meantime, the derivation and short -term culture of primary cells from solid tumors have thus gained significant importance in personalized cancer therapy and drug design.

Dissociated Tumor Cells (DTCs) are patient-derived, single cell suspension, obtained after dissociation of a solid tumor using enzymatic and/or mechanical approaches. DTCs recapitulate the cellular composition of the tumor microenvironment and thus are very important in the preclinical research, the field of personalized medicine and drug development. DTCs contain various cell populations present in the tumor microenvironment such as tumor cells, epithelial cells, fibroblasts, stromal and immune cells.

To create powerful tools for cancer research and drug discovery, FIDELIS RESEARCH offers the infrastructure for prospective collection of fresh tumor tissue on client demand as well as the subsequent preparation of variety of dissociated tumor cells, according to proven Standard Operating Procedures.

Tissues are obtained fully consented from approved medical centers following strict ethical standards.

Viable fresh tissue is collected and transported to Fidelis Research laboratories within two to four hours.

FIDELIS RESEARCH can provide to its clients various DTCs types (lung tumors, breast, kidney, ovarian, liver, colon, etc.). Clients can also request matched sets of viable peripheral blood mononuclear cells (PBMCs), normal tumor-adjacent tissue or tumor tissue.

TILs can be subsequently characterized at FIDELIS RESEARCH by flow cytometry.

Isolation of Human Cord Blood CD34+ Cells, Frozen

CD34 is a glycosylated transmembrane protein and represents a well-known marker for primitive blood- and bone marrow-derived progenitor cells. CD34+ stem cells are multipotent and can give rise to all cell types in blood. Additionally, CD34+ cells are responsible for all lymphohematopoietic lineages even though they comprise only a small percentage of the cell population.

FIDELIS RESEARCH offers primary human CD34+ cells isolated from umbilical cord blood mononuclear cells which include hematopoietic stem and progenitor cells. CD34+ cells are isolated from cord blood using positive immunomagnetic separation techniques, from single or mixed healthy donors. CD34+ progenitor cells are suitable for a series of studies for directed differentiation into more committed types of blood cells and endothelial lineages. Immediately after isolation, the freshly prepared CD34+ progenitor cells are cryopreserved using a serum-free freezing medium.

CD34+ cells are collected fully consented from approved medical centers following strict ethical standards and protocols approved by Local Ethics Committee or equivalent regulatory authority.

Donor options:

☐ Single

☐ Mixed

Human Cord Blood Mononuclear Cells, Frozen

FIDELIS RESEARCH offers primary mononuclear cells (MNCs), derived from the umbilical cord blood (CB), which include hematopoietic lineage cells such as lymphocytes, monocytes, stem and progenitor cells as well as mesenchymal stromal cells. These cells are critical components of the immune system and play key roles in the humoral and cell-mediated immune responses. Mononuclear cells are commonly used in various research fields and clinical applications in the areas of immunology, infectious disease, hematological malignancies, vaccine development, transplant therapy, and high-throughput screening. MNCs are isolated from CB using density gradient centrifugation protocol. This highly pure population of cells makes them ideal for further isolations of specific cell types within the mononuclear fraction.

Isolated cells are normally frozen in CryoStorTM CS10. We can also use freezing media as specified by the customer.

Human Cord Blood Plasma, Frozen

Human cord blood plasma contains a variety of cytokines, chemokines, and growth factors that affect the proliferation and function of immune cells. Recent studies have demonstrated a wide therapeutic potential of the human cord blood plasma for a broad range of diseases 1, 2. Human cord blood plasma begins to attract more and more the attention of the scientists. Some researchers believe that one day, proteins from the cord blood plasma may be able to help people with neurodegenerative diseases, like Alzheimer’s 3.

FIDELIS RESEARCH offers collection of human plasma isolated from cord blood using centrifugation. Collection of cord blood is a noninvasive and painless procedure performed at our partnering hospitals by our own procurement staff. Fresh whole cord blood, that is negative for HIV, HBV, and HCV, is collected in sterile collection bags containing the anticoagulant citrate phosphate double dextrose (CP2D) and transported to our local cell manufacturing facility where the blood is spun down.

Plasma is collected using consent forms and protocols approved by Local Ethics Committee and/or an equivalent regulatory authority.

 

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