Labor-Dienstleistungen

Cell culture refers to laboratory methods enabling the in-vitro growth of cells under temperature and nutrient controlled conditions. Cultured cells represent a very versatile tool when investigating basic scientific and translation research issues. What are considered excellent model systems to study cell physiology, biochemistry and genetics, cell cultures also play a key role in the production of viral vaccines, the development of new drugs and in toxicity assays.

Primary cultures are cells freshly isolated from a tissue, and preserved in-vitro under optimized conditions. When cultured, these cells are able to make a finite number of cell divisions, and then undergo senescence and stop dividing. Given that they derive directly from donor tissue, primary cells closely mimic the physiological state of cells in vivo and retain the main physiological characteristics of the source tissue. That is why they are considered a powerful in-vitro tool for the study of specific diseases.

We perform isolation of different cell types - epithelial cells, fibroblasts, keratinocytes, PBMCs, mesenchymal stem cells - from human and animal organs or tissues. What is more, we perform the expansion of in-vitro stabilized tumor cell lines, dissociated primary cell cultures (neuronal, glial, muscle, hepatic) and organotypic primary cultures (derived from the central nervous system, epithelial tissues and muscle).

From cell isolation and expansion to cryo-storage, the service includes:

● Acquisition of the tissue sample

● Enzymatic (trypsin, collagenase) or mechanical dissociation of the tissue

● Cell culture set up

● In vitro cell culturing

● Preparation, labeling and cryopreservation (if relevant)

● Delivery to customer or cryo-storage in our biorepository

Cell lines should be stored in nitrogen vapors in homogeneous batches, labeled with name, batch code and date of freezing. This way, the random quality controls on single tubes are representative of all samples belonging to the same batch.

Generating a cell bank is a crucial process in biological material production. The bioKryo Italy team, with its longstanding experience in this field, provides services for cell line expansion and preparation of master and working cell banks. This service will help streamline your testing and turnaround time, avoid serial banking, thus representing an ideal solution for your valuable human or animal cells; either generated from tissue or blood, or already established as cell lines (i.e., immortalized, stable-recombinant, tumor, iPS cell lines and stem cells).

The cell bank expansion and generation service includes:

Creation of master and/or working cell banks consisting of a number of labeled tubes according to the customer's indication (from 20 to 200 cryovials per cell line). Cryopreservation is performed using commercially qualified freezing media.

• Growth and expansion of the cell line to obtain the number of cells required to freeze the "master" batch.
• Thawing and expanding a tube from the "master" to obtain the number of cells required for the project ("working" batch).
• Cell viability assays following thawing and each passage coupled with acquisition of images of the cell morphology at different confluence.
• Cell growth curve monitoring and duplication time calculation.
• Sterility and identity tests (upon request).
• Delivery or cryo-storage of the cells controlled upon request in terms of viability, identity verification and absence of contamination.

Blood is made up of a suspension of cells, erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets) in a liquid (plasma) composed mainly of water, proteins, hormones, glucose and clotting factors. The serum, on the other hand, is what remains of the blood after having removed the cellular elements and the clotting factors.

Blood and its derivatives are the source of several molecules and analytes, such as genomic DNA, circulating nucleic acids, metabolites, hormones and cytokines. These elements are easily accessible and obtainable by patients and healthy individuals with non-invasive procedures (a simple blood draw) and provide valuable information for the biomedical, pharmaceutical, biotechnology and academic research communities.

bioKryo Italy performs the following blood component separation services:

• Plasma
  
  Plasma separation from peripheral blood collected with anticoagulants (e.g. EDTA, heparin) is performed by centrifugation. Approximately 2-4 ml of plasma is isolated from a 10 ml tube of whole blood, typically aliquoted in a volume of 1 ml and stored at –80 ° C.
• Serum
  
  Serum separation from peripheral blood collected without anticoagulants is performed by centrifugation. Approximately 2-4 ml of serum is isolated from a 10 ml tube of whole blood, typically aliquoted in a volume of 1 ml and stored at –80 ° C.
• Buffy Coat
  
  The buffy coat consisting of a mixture of lymphocytes, monocytes, granulocytes and platelets is isolated from peripheral blood by centrifugation. The isolated buffy coat is stored at –80 ° C.
• PBMCs
  
  Peripheral blood mononuclear cells contain mononuclear leukocytes such as lymphocytes (T cells, B cells, NK cells), monocytes, dendritic cells and basophils. PBMC isolation is performed by density gradient centrifugation (e.g. Ficoll TM). Following isolation, PMBC counts and viability are assessed for each isolation and the cells are cryopreserved in vapour of nitrogen.

The blood sample processing service includes:

● Rapid fractionation and isolation of blood components

● Viability assessment where necessary and upon request

● Preparation and labeling of cryogenic tubes containing blood components

● Delivery or storage of biological samples at different temperatures

● Characterization of the biological samples through different biochemical and molecular methods

For best results, blood samples should arrive in bioKryo Italy within two to four hours of collection and

processed within one day of receipt.

Rigorous standardization of biological material collection, processing and storage is essential to guarantee reproducibility and comparability of results obtained. In particular, to produce valid and robust biomolecular and omics data, researchers need to rely on high-quality nucleic acids.

bioKyo Italy performs nucleic acid purification services from a wide range of sample types, including whole blood, plasma, serum, buffy coat, tissue and in vitro cultured cells, using standardized commercial methods to ensure high purity and yield.

• DNA
  
  DNA isolation is a crucial step for all research laboratories relying on genomic technologies (e.g. next generation sequencing, PCR amplification and array-based genotyping analysis) for genetic studies of human diseases or for developing new biomarkers and diagnostic tools. We perform DNA purification services and, for genomic applications requiring high molecular weight genomic DNA, we also evaluate DNA integrity using a microfluidic platform for estimating the DIN (DNA Integrity Number).

• RNA
  
  RNA extraction represents a key step for research laboratories performing molecular and cellular biology studies that rely on genomic technologies such as real-time PCR, microarray gene expression analysis and next-generation sequencing (RNA-Seq). We perform RNA purification services and, upon request, evaluate RNA integrity using a microfluidic platform for estimating the RIN (RNA Integrity Number).

• microRNA
  
  microRNAs are small single-stranded non-coding RNA molecules acting as regulators of gene expression. microRNA expression analysis may provide important information on the interpretation of the pathogenesis of many human diseases. The altered expression of microRNAs in several pathological conditions makes them attractive as diagnostic and prognostic biomarkers, and as molecular targets for therapeutic purposes. We perform microRNA purification services from a wide range of sample types, including whole blood, plasma, serum, buffy coat, tissue and cultured cells.

The nucleic acid purification service includes:

• Extraction of nucleic acids from biological samples.
• Accurate quantification of purified nucleic acids by fluorimetric methods.
• Evaluation of the integrity of extracted DNA/RNA using a microfluidic platform for estimating the DIN/RIN (upon request).
• Preparation and labeling of the tubes.
• Delivery or storage.

Cell line authentication - STR profile

Cultured cells are valuable biomedical research tools. However, misidentification and cross-contamination of cell lines represent an ongoing problem that can jeopardize result validity and robustness. For that reason, scientific journals and funding agencies require researchers to ascertain that their cell lines are authentic. The analysis of STR (Short Tandem Repeat) polymorphic genetic markers represents the gold standard for establishing the identity of human samples. By providing a highly specific genetic fingerprint, STR profiling enables human cell lines to be uniquely identified, and to ensure that they are neither contaminated by other cells nor that they genetically differ from original stocks.

The STR profile-authentication service includes:

• Genomic DNA purification and quantification from live/frozen cells.
• PCR co-amplification and detection by capillary electrophoresis of nine autosomal STR molecular markers (D21S11, D7S820, CSF1PO, TH01, D13S317, D16S539, vWA, TPOX, D5S818) together with the sex-identifying amelogenin.
• Analysis of the obtained profiles.
• Complete description of the STR profile and analysis report including electropherogram

The STR profile is compared to the standard reference of the cell line to confirm its identity.

Microsatellite instability (MSI) test

Microsatellites are short, tandem repeated DNA sequences (one to six base pairs in length) distributed throughout the human genome. Microsatellite Instability (MSI) represents hypermutability in those sites due to impaired DNA mismatch repair (MMR). Genomic instability arises in a variety of human neoplasms and may be the first evidence of an MMR deficiency. MSI status is a predictive biomarker of a cancer positive response to immunotherapies such as immune checkpoint blockade inhibitors.

The MSI service is intended for research purpose and includes:

• Genomic DNA purification and quantification from live/frozen cells.
• PCR co-amplification and detection by capillary electrophoresis of five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide repeat markers (Penta C and Penta D).
• Analysis of the obtained profiles. The presence of numerous extra alleles in the microsatellite profile indicates genome instability.
• Full description of the MSI profile and analysis report including electropherogram

Contamination of cell cultures with bacteria, fungi and mycoplasma is a widespread laboratory phenomenon that can jeopardize experimental result reproducibility by altering cellular parameters such as metabolism, growth and vitality. Viral contamination may arise from the original source of derived cell lines or from adventitious introduction of viruses during manufacturing processes.

Cell culture contamination cannot always be easily detected and can be highly insidious. Contaminants often remain undetected until the cells show evident signs of morphological alterations. Consequently, cell cultures should be regularly monitored with sensitive methods to detect contamination in the early phases.

Bacteria/ Fungi 

We perform sterility tests by direct inoculation of cell culture supernatant on solid medium for detecting bacteria and fungi. Specifically, the analysis is carried out in duplicate for each sample on Columbia Agar supplemented with five percent Sheep Blood plates for the detection of bacteria and Sabouraud Dextrose Agar (SDA) for fungi. Reference strains for bacteria and fungi are used according to the European Pharmacopoeia (EP 2.6.1).

Mycoplasma

Mycoplasma are very small prokaryotic organisms that cannot be detected by microscopic inspection and may remain hidden in cell cultures for long periods. Therefore, routine screening of cell lines for mycoplasma contamination is of paramount importance. We perform the mycoplasma analysis on the supernatant of cell cultures based on two different types of tests:

• endpoint PCR targeting highly conserved 16S rRNA coding region of the mycoplasma genome allowing the detection of a wide range of Mycoplasma species,
• quantitative PCR (qPCR) targeting a highly conserved region within the mycoplasma genome to detect all species included in the European Pharmacopoeia Chapter 2.6.7 (EP 2.6.7).

Viral Screening

We perform analysis for detecting human immunodeficiency virus (HIV), hepatitis C (HCV), hepatitis B (HBV) and cytomegalovirus (CMV) by RT-PCR analysis following purification of nucleic acids viral from cultured cells. 

The service of analysis of cell contamination includes:

• Verification of absence of contamination from bacteria, fungi, mycoplasma and viruses in cell lines,
• Full interpretation of results by a specialist,
• Analysis report for each analyzed cell line.
  
   

Cultured cells may acquire genomic abnormalities due to long-term in vitro growing or genetic manipulations (i.e., the introduction of exogenous DNA during the reprogramming or CRISPR-Cas9 editing processes). Alterations of the cellular genomic structure can drastically modify the cellular characteristics, and thus impairing result robustness in downstream research experiments. For a reliable use of cell cultures, it is therefore necessary to conduct periodic monitoring to check for genomic abberations. Thanks to our operators’ longstanding specialized experience in cell genomic control, bioKryo Italy provides cytogenetic services aimed at confirming the chromosomal structural and numerical stability of research cell lines. Our analyses are performed on different types of cells, such as stabilized cell lines, embryonic stem (ES), induced pluripotent stem (iPS), somatic and tumor cell lines. The tests are performed for research purposes only and not for diagnostic or prenatal screening analyses.

Q-banding Karyotype

Karyotype analysis is a conventional chromosomal banding technique that represents the gold standard test for detecting genetic abnormalities in cell cultures. It makes it possible to evaluate the number and structure of chromosomes to identify rearrangements (inversions, duplications/deletions, balanced and unbalanced translocations, aneuploidy) with a resolution > 5-10 Mb and mosaicism > ten percent. We perform karyotype analysis by the Q-banding method involving use of fluorescent dye Quinacrine. Our qualified fully equipped fluorescence microscope and GenASIs Bandview software module for karyotyping provides accurate, repeatable and standardized analyses. 

Spectral Karyotype- SKY

In the event of complex interchromosomal rearrangements, spectral karyotype (SKY) technique allows the detection of cryptic or submicroscopic genetic anomalies that cannot be resolved with conventional cytogenetics. This method is based on the hybridization of probes labeled with distinct fluorescent dyes in different combinations in order to generate spectrally different colors for each chromosome.

❖ With both Q-banding and SKY, chromosomes are classified according to the ISCN 2016 guidelines: An International System for Human Cytogenomic Nomenclature

The Q-banding/spectral (SKY) karyotype service includes:

● Thawing and expansion of cells (both human and murine)

● Spreading of chromosome metaphase and number/quality evaluation

● Staining for Q-banding/hybridization for SKY

● Analysis of at least 20 Q-banded metaphases for each cell line

● At least one high-quality image of a representative karyotype in a publication ready format

● Comprehensive analysis of results and interpretive consultation by a specialist

● Report of analysis for each analyzed cell line

Molecular Karyotype - aCGH

The high resolution molecular karyotype based on array platforms enables the detection of aberrations and rearrangements that would be overlooked with traditional chromosome investigations. As an example, Copy Number Variations (CNVs) or heterozygous loss conditions (LOH) can be characterized using array-based comparative genomic hybridization (aCGH). This method is able to identify genomic anomalies as small as a few thousand base pairs, and precisely define the altered genomic region and genes involved.

The whole service from live or frozen cell cultures includes:

• Thawing and expansion of cells.
• Purification and quantification of genomic DNA.
• DNA labeling and hybridization with Agilent platform.
• Data extraction and analysis with Agilent Cytogenomics software.
• At least one high-quality image in a publication-suitable format.
• Comprehensive analysis of results and interpretive consultation by a specialist.
• Report of analysis for each analyzed cell line including raw data.
  
   

The discovery of induced pluripotent stem cells (iPSCs) has revolutionized research based on personalized medicine. IPSCs are a type of pluripotent cells that can be generated from highly differentiated cells (typically fibroblasts and PBMCs) by introducing multiple pluripotency factors. This technology allows researchers to obtain cells in an embryonic-like stage with the capacity to undertake multiple differentiation destinies without embryonic materials and associated ethical concerns.

iPSCs present a promising alternative source of patient-specific stem cells with great potential for therapeutic developments in the field of regenerative medicine. They are also an excellent experimental platform for disease modeling, drug screening and toxicology studies.

The process of generating iPSCs properly reprogrammed in terms of pluripotency features and genomic stability is both time consuming and requires extensive technical experience.

Generation of hiPSCs from adult somatic cells

We offer the hiPSC generation services from healthy donor or patient-derived fibroblasts and PBMCs by employing advanced iPSC reprogramming factor delivery using both episomal vectors and Sendai virus. Once generated, the iPSCs are tested in terms of pluripotency and their ability to differentiate into the three embryonic layers (trilineage differentiation). Once generated, the cells are also assessed for genomic stability and the absence of fungal/bacterial/mycoplasma contamination. 

The cell reprogramming service includes:

• Thawing, recovery and culturing fibroblasts or PBMCs.
• Transfection/transduction of reprogramming factors.
• Selection, isolation and expansion of single generated iPSC colonies.
• Evaluation (morphology, pluripotency, trilineage differentiation) and expansion of three iPS clones for each cell line
• Evaluation of the absence of transduction vectors
• Sterility test for bacteria and fungi in each clone produced
• Analysis of mycoplasma by PCR in each generated clone
• Cryopreservation of at least five cryogenic tubes for each clone (> 1x106 cells/tube)
• Post-thaw viability assay of each clone
• Delivery or cryo-storage in our facility 
  
   

Induced pluripotent stem cells (iPSCs) are able to differentiate into other cell types, and self-renew indefinitely without senescence. As a result, they represent a virtually unlimited source of diverse cell types, including cells difficult to obtain from primary tissues (such as cardiomyocytes, neurons, and megakaryocytes). Furthermore, they represent a powerful tool for developing highly physiological and patient-specific study models. bioKryo Italy operators have collaborated on several studies on stem cells and iPSCs, and make their experience available for customizable differentiation services of iPSCs into different cell types.

Cardiomyocyte differentiation of hiPSCs   

We apply a validated and robust protocol so as to perform the differentiation of iPSCs into functional cardiomyocytes that can be used as in vitro models for electrophysiological studies, biochemical analyses, the development of new drugs, toxicity screening and genetic studies. Cardiomyocytes obtained in this way, represent a valid alternative to embryonic stem cells and animal models. We generate iPSC-derived cardiomyocytes from healthy donors or individuals with medical conditions.

The whole service includes:

• Thawing, expansion and validation of iPSCs.
• Cardiomyocyte differentiation of iPSCs.
• Phase contrast images of parental iPSC lines following expansion and differentiation into cardiomyocytes.
• Video recording the pulsating activity of cardiomyocyte culture.
• Characterization of differentiated cells by immunocytochemical staining with three specific markers (cTNT, SMA, Nkx2.5).
• Delivery of functional cardiomyocytes and analysis report for each analyzed cell line.

Megakaryocyte differentiation of hiPSCs

Megakaryocytes (MKs) are highly specialized hematopoietic cells responsible for producing and releasing platelets that play an essential role in primary haemostasis and blood clotting. MKs are myeloid cells representing only approx. one percent of the cell population residing in bone marrow. The ex vivo generation of MKs from iPSC provides an unlimited source of platelets that can be cryo-stored and made available on demand with no need to perform frequent patient blood draws.

By means of a validated and robust protocol, we perform the differentiation of iPSCs into functional megakaryocytes that can be used as in vitro models for biochemical and pharmacological assays. We generate iPSC-derived megakaryocytes from healthy donors or individuals with medical conditions.

The whole service includes:

• Thawing, expansion and validation of iPSCs.
• Megakaryocyte differentiation of iPSCs.
• Phase contrast images of parental iPSC lines following expansion and differentiation into megakaryocytes.
• Characterization of differentiated cells by flow cytometry for the expression of specific megakaryocyte surface marker CD41 and for evaluation of ploidy.
• Delivery of functional megakaryocytes and analysis report for each analyzed cell line.

Trilineage differentiation of hMSC

Human mesenchymal stem cells (hMSCs), also called multipotent stromal cells, represent an example of adult stem cells. Besides bone marrow, hMSCs can be isolated from a wide variety of other tissues, such as adipose tissue, cord blood, placenta, skin, and hair follicles. hMSCs harbor great potential in tissue regeneration and cell therapy owing to their ability to produce a large number of specialized cells including adipocytes, osteocytes, chondrocytes, epithelial cells, neuron-like cells and hepatocytes. As well as isolation from tissues, the use of hMSCs requires confirmation of their stem potential, i.e. rigorous verification of their ability to differentiate into adipocytes, chondrocytes and osteoblasts when cultured in vitro (trilineage differentiation).

The whole service includes:

• Thawing and expansion of hMSCs from tissues of different origins.
• Morphology monitoring.
• Evaluation of trilineage differentiation by staining with:
  
  ○  Alizarin Red to identify the presence of mineralized nodules and calcium deposition that are specific features of osteogenic differentiation.
  
  ○ Oil Red to reveal triglyceride deposits, the main components of the lipid deposits present in adipocytes.
  
  ○ Alcian Blue to highlight the formation of cartilage aggregates and the accumulation of proteoglycans.
• Analysis of the expression levels of specific markers by qRT-PCR.
• Delivery of functional differentiated cells and analysis report for each analyzed cell line.

Generation and tissue-specific differentiation  of organoids/spheroids

Cell cultures are essential tools for basic, translational and preclinical research. In addition to the classic cell culture model, which provides for the in-vitro growth of cells in monolayer (2D), 3D cell growth systems have attracted much attention in recent years. 3D in vitro systems allow the formation of cellular aggregates, the so-called organoids (or spheroids). These structures more faithfully reproduce the architecture of human organs and tissues than cell monolayers, and thus represent promising models for studying physiological processes. Furthermore, organoids are amenable to all cellular/molecular/pharmacological analyses already developed for traditional cell lines. Their resemblance to human organs means that the study of organoids and spheroids harbors great potential for translational research.

From human induced pluripotent stem cells (hiPSCs), we generate human brain organoids that are able to recapitulate the characteristics of human cortical development.

The whole service includes:

• Thawing, expansion and validation of iPSCs.
• Tissue-specific differentiation of iPSCs.
• Phase contrast images of both iPSC lines following expansion and organoids in different stages of growth.
• Characterization with specific markers through immunofluorescence staining.
• Delivery of functional organoids.