Cancer Stem Cells (CSCs) are a subpopulation of tumor cells that have the ability of self-renew, differentiation, and initiation of new tumors. They are resistant to standard cytotoxic chemotherapy and ionizing radiation, and are common cause for metastases formation. Therefore, this makes them an important target for the development of novel therapies. Cancer stem cells have been identified and isolated from many solid tumors, including - brain, breast, lung, colon etc. It is shown that when grown in non-adherent serum-free environment CSCs have the ability to form multicellular three-dimensional spheres (tumorspheres) in vitro.

Tumorsphere cultivation is based on culturing cancer cells onto ultralow attachment surface in serum-free media under the supplementation with growth factors. This method is widely used to analyze the self-renewal capability of CSCs and to enrich these cells from bulk cancer cells. It also provides a reliable platform for screening potential anti-CSC agents. The in vitro anti-proliferation activity of potential agents selected from tumorsphere assay is more translatable into in vivo anti-tumorigenic activity compared with general monolayer culture. Tumorsphere assay can also measure the outcome of clinical trials for potential anti-cancer agents. In addition, tumorsphere assay may be a promising strategy in the innovation of future cancer therapeutics and may help in the screening of anti-cancer small-molecule chemicals.

Fidelis Research strives to offer a wide variety of 3D tumorsphere cultures composed of cancer stem cells isolated from broad assortment of solid tumors.

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.

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.

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.

Immortalized cancer cell lines for decades have been an exciting subject of study on tumor biology. They were used for antitumor activity testing of a number of anticancer therapeutics. The problem with immortalized cell lines is that they rarely represent faithfully the tissue from which they are sourced from. Thus, research on them cannot always predict the actual behavior of the tumor. Mainly for this reason, it is increasingly favored to use primary tumor cell cultures obtained directly from the patient's tissue.

The cell cultures from primary tumors and metastases of different kind of cancers represent a valuable tool used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging, signaling studies), the effects of drugs and toxic compounds on the cells and mutagenesis and carcinogenesis. It is also used in drug screening as well as for the development of biological compounds (such as: vaccines, therapeutic proteins) on a large scale. Primary cancer cultures currently begin to constitute the golden standard of in vitro model, therefore their implementation into laboratory practice is highly recommended.

Fidelis Research offers primary human cancer cell cultures, developed from fresh samples of a range of tumor tissue types (lung, breast, kidney, ovarian, liver, colon tumors, etc.), which represents improved models for cancer research. Our product is accompanied with the detailed information about the pathological diagnoses and key mutations, which can elucidate the defined mechanisms behind cancer formation.

Benefits of using primary cancer cell cultures:

• These cells are heterogeneous population with various genetic origin
• Entire pathology information available for each batch of cells
• They are representative for the tumor type from which they were sourced
• Suitable for molecular studies and genetic manipulation

Applications of primary cancer cell cultures:

• General research for understanding cancer biology: They can be used to study the mechanism and cause of cancer and the altered signaling pathways.
• Research and development of new drugs and therapies.
• Testing of drug cytotoxicity: They are useful for studies on the effect and safe drug dosage, and/or drug carriers (nanoparticles).

Immunomagnetic separation is an array of methods relying on antibody binding to enrich certain cell types within a suspension. The aim is to facilitate isolation of desired cell phenotypes compared to other methods such as density gradient centrifugation. The method relies on the interactions between a cell surface molecule and its ligand, immobilized on a magnetic particle, while the sample is being incubated in a magnetic field. Depending on the type of molecule used for binding, immunomagnetic separation methods can be grouped into positive and negative selection.

Positive selection immunomagnetic cell separation methods involve directly labeling desired cells for selection with an antibody or ligand that targets a specific cell surface protein:

• Isolated cells are highly purified.
• Isolated cells are usually bound by antibodies and magnetic particles.
• Antibody cocktail targets a unique surface marker on the target cells, removing them from the suspension.
• Additional cell populations from the negative fraction can be isolated.

Negative selection immunomagnetic cell separation methods involve labeling unwanted cell types for removal with antibodies or ligands targeting specific cell surface proteins:

• Antibody cocktail targets all unwanted cells, removing them from the suspension when a magnetic field is applied, leaving behind the desired cell population.
• Protocols are faster and easier with minimal sample manipulation.
• Isolated cells are not bound by magnetic particles.

Applications

In general, the tradeoff between both types of immunomagnetic separation is best defined as precision of isolation (positive selection) versus gentle treatment of the cells (negative selection). For most applications, labeling cells with antibodies and magnetic particles during positive selection does not interfere with downstream assays, leaving positive selection as the preferred method to isolate a strictly defined target cell population. However, it should be taken into consideration if the specific research application requires unlabeled cells, which may be important when:

• Antibodies bound to a cell surface antigen is known to cause unwanted intracellular signalling.
• Antibodies or magnetic particles bound to cell surface proteins affect downstream use of the cells.
• Isolating “untouched cells”, i. e. those isolated directly from target organs. In the context of immune cells, negative selection allows for the isolation of non-activated cells.
• Trying to reduce fluorescence-activated cell sorting (FACS) running time by pre-enriching the target cell population. Negative selection is the preferred choice in order to avoid labeling which may interfere with the FACS procedure. This can be done upstream of FACS for a wide variety of specific cell subsets, including antigen-specific cells, subsets of T helper cells, B cells, and innate lymphoid cells (ILCs).

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 offer 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.

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:

• Isolation of miRNA;
• Isolation of ccfDNA;
• Isolation of ccfDNA and ccfRNA ;
• Isolation of genomic DNA;
• Isolation of total RNA;

Human T Cell expansion is a method of inducing the proliferation of T-cells in culture after activating them. It has become an increasingly important tool in the development of both autologous and allogeneic immunotherapies, as T cells emerge as an exciting option to treat a wide range of diseases. The method relies on microspheres (beads) loaded with anti-CD3/CD28 antibodies in order to mimic activation of T cells by antigen-presenting cells, after which the cells are also exposed to Interleukin 2 in order to trigger their rapid proliferation. Typically, the cells are incubated with CD28/CD3 beads immediately after putting them into culture in a serum-free medium and then also treated with IL-2 over the course of 14 days.

Applications

This simple, efficient and consistent technology out-performs and replaces traditional home-brew methods for generic activation based on antigen-presenting cells (APCs), mitogens (e.g. ConA, PHA), soluble/plate-bound antibodies, or chemical activators, and is well documented in published literature. Adoptive T cell therapy is a rapidly progressing field, and optimized protocols for the scalable manufacture of T cells are essential to maximize the therapeutic potential of patient-derived T cells, with a single administration requiring billions of expanded T cells. Expansion and re-infusion of genetically engineered T cells has the potential to revolutionize the treatment of leukemia and related blood cancers. Since activated T cells can also be used in clinical research and undergo any downstream processing, such as cytokine analysis or immunoprecipitation, and can be transfected with high efficiency, the method finds an application in the clinical study of the immune response in various diseases. Particularly sought-after T-cell lines to expand for such purposes include antigen-specific T cells as well as CD3+CD56+ NKT cells.

Fidelis Research aims to provide clonally expanded T cells from peripheral blood of patients with various indications for a wide array of research applications.

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 disease-state and control PBMCs.

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.

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' tеam guarantees highly professional sample collection and further processing.

Fidelis has a dedicated sample storage and management facility, offering both long- and short-term sample storage services to ensure sample integrity. The temperatures in which we can store the samples cover the full range of sample management needs:

• Ambient
• 2-8° C
• - 20° C
• - 80° C
• - 150° C

Our storage facility is equipped with a back-up electricity generator which will keep the freezers running even in the situation of electricity shutdown. We have real-time remote temperature monitoring and alert system providing immediate signal in the case of temperature deviations in any of the freezers.

Fidelis’ inventory management allows for visibility and control of our customers’ samples and ensures fast and flexible data reporting, shipping requests and search options.

In addition, our team has the capabilities to provide retrieval and transportation of samples from collection sites, client-specified locations or other point of receipt to our storage facility, as well as specialized shipping of small or large batches of samples to clients’ locations globally.