• MeSH
• Cells, Cultured MeSH
• Neoplasms MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• onkologie

• MeSH
• Dendritic Cells MeSH

• Conspectus
• Buněčná biologie. Cytologie
• NML Fields
• cytologie, klinická cytologie
• biologie

Natural killer (NK) cells constitute the predominant innate lymphocyte subset that mediates the anti-viral and anti-tumor immune responses. NK cells use an array of innate receptors to sense their environment and to respond to infections, cellular stress and transformation. The resulting NK cell activation, including cytotoxicity and cytokine production, is a fundamental component of the early immune response. The most recent discoveries in NK cell biology have stimulated the translational research that has led to remarkable results for the treatment of human malignancies. Therefore, the rapid isolation of NK cells from the peripheral blood or tumor microenvironment and the subsequent assessment of cytolytic function are crucial to the study of their potency and NK cell-mediated immunosurveillance. Here, we provide protocols for NK cell isolation and the assessment of NK cell cytotoxicity using flow cytometry.

• MeSH
• Lymphocyte Activation MeSH
• Killer Cells, Natural immunology   MeSH
• Cytotoxicity, Immunologic * MeSH
• Cytotoxicity Tests, Immunologic methods   MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Cell Separation methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bone marrow is an important source of mesenchymal stem cells (MSCs), and a promising tool for cytotherapy. MSC utilization is limited by low cell yields obtained under standard isolation protocols. Herein, used bone marrow collection sets were evaluated as a valuable source of MSCs. Adherent cells washed from the collection sets were examined for widely accepted criteria defining MSCs. Significant numbers of cells (median 9million per set in passage 1) with colony-forming activity and high proliferative potential at low seeding densities were obtained. These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29) and negative for most haematopoietic and endothelial cell markers (CD45, CD34, CD11a, CD235a, HLA-DR, CD144). The cells were capable of differentiation along adipogenic, osteogenic and chondrogenic pathways. Washing out bone marrow collection sets may constitute a highly ethical source of MSCs for research purposes and may be utilized also in clinical applications.

• MeSH
• Adipogenesis MeSH
• Biomarkers metabolism   MeSH
• Cell Adhesion MeSH
• Cell Culture Techniques methods   MeSH
• Cell Lineage MeSH
• Bone Marrow Cells cytology   MeSH
• Chondrogenesis MeSH
• Financing, Organized MeSH
• Humans MeSH
• Mesenchymal Stem Cells cytology   MeSH
• Antibodies, Monoclonal MeSH
• Osteogenesis MeSH
• Cell Count MeSH
• Cell Proliferation MeSH
• Cell Separation MeSH
• Cell Shape MeSH
• Check Tag
• Humans MeSH

Successful isolation of Langerhans islets is a crucial prerequisite for their experimental or possible clinical use such as transplantation. Centrifugation in a Ficoll gradient is a common step used for separation of Langerhans islets from exocrine tissue. However, islets have been reported to be negatively affected by employing Ficoll gradients. Therefore, the aim of this study was to modify the isolation procedure by excluding Ficoll gradient centrifugation to obtain a similar or better yield of viable, functional islets. In our modification of the isolation procedure, the separation of islets from exocrine tissue was based on their sedimentation rate combined with their differential ability to attach to the surface of culture dishes for suspension cells. The resulting purity of islets facilitated their handpicking from the suspension. The mean yield was 900 viable, insulin-producing islets per mouse, which was comparable to or even higher than the yield in commonly used protocols. Our modification of the isolation method may be useful when centrifugation in Ficoll gradient is undesirable due to potential toxicity.

• MeSH
• Cell Adhesion MeSH
• Islets of Langerhans cytology   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Inbred ICR MeSH
• Mice MeSH
• Pancreas MeSH
• Cell Count MeSH
• Cell Separation methods   MeSH
• Islets of Langerhans Transplantation methods   MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Cytological Techniques MeSH
• Molecular Biology methods   MeSH
• Image Cytometry * methods   MeSH
• Flow Cytometry * methods   MeSH
• Cell Separation methods   MeSH
• Publication type
• Laboratory Manual MeSH

• Conspectus
• Buněčná biologie. Cytologie
• NML Fields
• cytologie, klinická cytologie

Úvod: Zubní dřeň představuje relativně snadno dostupný zdroj mezenchymových kmenových buněk. Kmenové buňky ze zubní dřeně (KBZD) byly poprvé izolovány v roce 2000 a od té doby jsou intenzivně studovány. Cíl: Cílem studie bylo optimalizovat proces izolace KBZD, zejména zkrátit dobu, po kterou je zubní dřeň enzymaticky štěpena. U KBZD izolovaných novým postupem stanovit proliferační aktivitu, fenotyp, viabilitu a ověřit schopnost jejich diferenciace ve zralé buněčné typy – osteoblasty, chondroblasty a adipocyty. Materiály a metodika: Extrahovali jsme celkem pět stálých zubů a izolovali pět buněčných linií, které jsme kultivovali v modifikovaném kultivačním médiu (α-MEM) pro adultní mezenchymové progenitorové buňky, obohaceném o 2 % fetálního bovinního séra, růstové faktory, antibiotika, antimykotika, a doplněném o Insulin-Transferrin-Sodium-Selenium supplement. Pro stanovení viability, počtu a velikosti buněk jsme použili přístroje Vi-Cell analyzer a Z2-Counter. Fenotypová analýza byla provedena pomocí průtokového cytometru Cell Lab Quanta. Pro diferenciaci v chondroblasty, osteoblasty a adipocyty jsme použili komerčně dostupná diferenciační média. Průkaz diferenciace jsme dokazovali imunohistochemicky (osteokalcin a kolagen typu II) a histologickým barvením (modrý Massonův trichrom, barvení dle Kossy a olejová červeň). Výsledky: KBZD jsme kultivovali do 8. pasáže. Buňky dosáhly průměrně 47,8 ± 2,0 populačních zdvojení (angl. population doublings; PD). Průměrný čas potřebný pro zdvojení populace (angl. doubling time; DT), činil 39,2 ± 6,1 hodin. Po celou dobu kultivace byly buňky proliferačně aktivní. Průměrná viabilita v 2. pasáži byla 92,3 ± 1,5 % a v 8. pasáži 92,4 ± 1,4 %. Fenotypovou analýzou jsme prokázali vysokou expresi povrchových antigenů pro mezenchymové kmenové buňky (angl. cluster of differentiation; CD) CD13, CD29, CD44, CD90, pro tzv. „stromal associated“ znaky CD73, CD166, a naopak nízkou či nízce pozitivní expresi znaků CD31, CD34, CD45, typických pro endoteliální a hematopoetickou buněčnou řadu. KBZD se diferencovaly ve zralé buněčné typy, osteoblasty a chondroblasty. I přes proadipogenně silně působící médium buňky nediferencovaly v adipocyty. Závěr: Podařilo se nám optimalizovat izolační protokol pro KBZD tím, že jsme zkrátili dobu, po kterou je zubní dřeň enzymaticky štěpena. KBZD izolované touto metodou prokazovaly po celou dobu kultivace vysoký proliferační a diferenciační potenciál. Nezpozorovali jsme žádné známky spontánní diferenciace či degenerace. Právě pro vysokou proliferační aktivitu, široký diferenciační potenciál a snadnou dostupnost představují KBZD budoucnost v regenerativní medicíně.

Introduction: The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). They were isolated for the first time in 2000 and since then many researchers have investigated and analysed their biological characteristics. Aim: The purpose of this study was to optimize the isolation protocol for DPSCs, namely to shorten the time of enzymatic digestion of the dental pulp, and to cultivate isolated DPSCs using this new approach, investigate their proliferation, phenotype, cell viability and determine their ability to differentiate into mature cells, chondroblasts, osteoblasts, and adipocytes. Materials and methods: Out of five extracted permanent teeth, we isolated five dental pulp stem cell lineages. They were cultivated in a modified cultivation media (α-MEM) for mesenchymal adult progenitor cells containing 2 % fetal bovine serum (FBS) and supplemented with growth factors, antibiotics, antimycotics and Insulin-Transferrin-Sodium-Selenium supplement (ITS). The cell viability, cell count and other properties were examined using a Vi-Cell analyzer and Z2-Counter. The phenotype analysis was performed using a flow cytometer Cell Lab Quanta. For differentiation in chondroblasts, osteoblasts and adipocytes, we used commercially available differentiation media. The evidence of differentiation was proved by the immunocytochemistry (osteocalcin and collagen type II) and histological staining (blue Masson's trichrome, von Koss stain and oil red). Results: We were able to cultivate DPSCs over 47.8 ± 2.0 population doublings (PD). The average population doubling time (DT) was 39.2 ± 6.1 hours. The average cell viability was 92.3 ± 1.5 % in the second passage and 92.4 ± 1.4 % in the eighth passage. DPSCs showed high positivity for mesenchymal stem cell markers (cluster of differentiation; CD) CD29, CD44, CD90 and for stromal associated markers CD13, CD73, CD166 and negative expression or low positivity for hematopoietic markers CD34, CD45 and for CD31. DPSCs differentiated into osteoblasts and chondroblasts. Even after the exposition of the strong adipogenic medium they did not show any signs of differentiation into adipocytes. Conclusion: We have successfully optimized the isolation protocol for DPSCs by shortening the time of enzymatic digestion of the dental pulp. DPSCs isolated using the new method demonstrated the high proliferation and differentiation potential throughout long-term cultivation. We did not observe any signs of spontaneous differentiation or cell degeneration. DPSCs seems to be the promising future for a regenerative and reparative medicine thanks to their remarkable high proliferative potential and ability to differentiate into many mature cell populations.

Summary: Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflick’s limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60–90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflick’s limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

• MeSH
• Bone Marrow Cells cytology   physiology   MeSH
• Femur cytology   physiology   growth & development   MeSH
• Research Support as Topic MeSH
• Cells, Cultured cytology   MeSH
• Humans MeSH
• Mesenchymal Stem Cells cytology   physiology   MeSH
• Flow Cytometry methods   utilization   MeSH
• Donor Selection methods   utilization   MeSH
• Dental Pulp cytology   growth & development   MeSH
• Tooth cytology   physiology   growth & development   MeSH
• Check Tag
• Humans MeSH

Oligodendrocyte progenitor cells (OPCs) play a pivotal role in both health and disease within the central nervous system, with oligodendrocytes, arising from resident OPCs, being the main myelinating cell type. Disruption in OPC numbers can lead to various deleterious health defects. Numerous studies have described techniques for isolating OPCs to obtain a better understanding of this cell type and to open doors for potential treatments of injury and disease. However, the techniques used in the majority of these studies involve several steps and are time consuming, with current culture protocols using serum and embryonic or postnatal cortical tissue as a source of isolation. We present a primary culture method for the direct isolation of functional adult rat OPCs, identified by neuron-glial antigen 2 (NG2) and platelet derived growth factor receptor alpha (PDGFrα) expression, which can be obtained from the adult spinal cord. Our method uses a simple serum-free cocktail of 3 growth factors - FGF2, PDGFAA, and IGF-I, to expand adult rat OPCs in vitro to 96% purity. Cultured cells can be expanded for at least 10 passages with very little manipulation and without losing their phenotypic progenitor cell properties, as shown by immunocytochemistry and RT-PCR. Cultured adult rat OPCs also maintain their ability to differentiate into GalC positive cells when incubated with factors known to stimulate their differentiation. This new isolation method provides a new source of easily accessible adult stem cells and a powerful tool for their expansion in vitro for studies aimed at central nervous system repair.

• MeSH
• Antigens metabolism   MeSH
• Platelet-Derived Growth Factor metabolism   MeSH
• Adult Stem Cells cytology   metabolism   MeSH
• Fibroblast Growth Factor 2 metabolism   MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Rats MeSH
• Spinal Cord cytology   metabolism   MeSH
• Oligodendroglia cytology   metabolism   MeSH
• Rats, Sprague-Dawley MeSH
• Proteoglycans metabolism   MeSH
• Cell Separation * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Úvod: Cílem práce bylo zavedení a optimalizace nového separačního protokolu pro kontinuální separaci mononukleárních buněk (CMNC) Spectra Optia v. 11 při přípravě štěpů PBPC pro autologní transplantace u pacientů s hematoonkologickými maligními onemocněními. Vyhodnocovali jsme výsledky 159 separací PBPC u: a) jen dobře mobilizovaných pacientů s předseparační koncentrací CD 34+ buněk v krvi vyšší než 20 v μl; b) pouze prvních separací provedených buď za pomoci CMNC Spectra Optia v. 11, nebo na přístrojích Cobe Spectra v. 6, v. 7; c) za použití standardního a velkoobjemového (LVL) režimu; d) u následně transplantovaných 56 pacientů jsme sledovali dobu přihojení štěpu v počtu neutrofilních leukocytů a trombocytů. Výsledky: U 52 pacientů separovaných v standardním režimu byl obsah CD 34+ buněk v přípravcích vysoký a nebyly nalezeny významné rozdíly mezi počtem CD 34+ buněk připravených na přístrojích Spectra Optia 8,6 (1,3–41) × 106 a Cobe Spectra 10,9 (1,8–45,6) × 106/kg příjemce (a = 0,05; p = 0,619). Při hodnocení vztahu výtěžku CD 34+ buněk a jejich předseparační koncentrace v krvi byly hodnoty korelačního koeficientu vysoké; u CMNC Spectra Optia R = 0,95 a u Cobe Spectra R = 0,93. U 107 pacientů, u nichž se prováděly LVL, nebyly nalezeny významné rozdíly mezi obsahem CD 34+ buněk připravených v LVL režimu na přístrojích Spectra Optia 10,9 (2–61,2) × 106 a Cobe Spectra 9,3 (2,4–86) × 106/kg příjemce (a = 0,05; p = 0,35). Při hodnocení vztahu výtěžku CD 34+ buněk a jejich předseparační koncentrace v krvi byly hodnoty korelačního koeficientu u CMNC Spectra Optia R = 0,93 a u Cobe Spectra R = 0,78. Z výsledků lze uzavřít, že hodnota předseparační koncentrace CD 34+ buněk v krvi je významným ukazatelem účinnosti mobilizace a lze z ní odhadnout pravděpodobný výtěžek buněk ze standardní a LVL separace. U LVL separací byl medián ztráty trombocytů významně nižší u CMNC Spectra Optia (45 %) než u Cobe Spectra (57 %). U 12 pacientů byla provedena autologní transplantace s podáním PBPC připravených v standardním režimu, zatímco ostatním 44 pacientům byly podány buňky připravené technikou LVL. Medián doby přihojení v počtu neutrofilních leukocytů a trombocytů u přípravků ze standardního režimu byl u CMNC Spectra Optia a Cobe Spectra 11 dnů, v počtu trombocytů u CMNC 14 dnů a u Cobe Spectra 12 dnů. U přípravků z LVL režimu byl medián doby přihojení v počtu neutrofilních leukocytů a trombocytů u PBPC u obou přístrojů shodný a činil 11 dnů a 13 dnů. Závěr: CMNC protokol Spectra Optia v. 11 je moderní, účinný a bezpečný systém, který vyhovuje pro provádění jak standardních tak LVL separací. U dobře mobilizovaných pacientů lze pomocí nového protokolu CMNC připravit potřebnou dávku CD 34+ buněk z jedné standardní nebo z jedné LVL separace. V průběhu výkonů jsme nezaznamenali žádné závažné nežádoucí reakce.

Background: The aim of the study was to evaluate and optimize the new protocol for continuous mononuclear cell collection (CMNC) Spectra Optia v. 11 (Terumo) which was used for PBPC collections in patients with haemato-oncological diseases. The results of 159 autologous PBPC collections were evaluated in the case of: (a) well mobilized patients with pre-collection CD 34+ cell concentration in blood higher than 20/μl, (b) only the first collections performed either using CMNC Spectra Optia v. 11 or Cobe Spectra v. 6, v. 7, Terumo (c) collections were performed in the Standard and Large volume Leukapheresis regimen, LVL. Engraftment data from 56 transplanted patients were assessed. Results: Standard collections were performed in 52 patients. The yield of CD 34+ cells was high, and no significant differences were found between the numbers of CD 34+ cells prepared using Spectra Optia 8.6 (1.3–41) × 106 and Cobe Spectra 10.9 (1.8–45.6) × 106 /kg b. w. (a = 0.05; p = 0.619). The dependence of CD 34+ cell yield on the pre-collection concentration of CD 34+ cells in blood can be considered as linear with high correlation coefficients in CMNC Spectra Optia R = 0.95, and Cobe Spectra R = 0.93. LVL collections were performed in 107 patients and there were no significant differences between the numbers of CD 34+ cells prepared using CMNC Spectra Optia 10.9 (2-61.2)× 106 and Cobe Spectra 9.3 (2.4-86) × 106 /kg b.w. (a = 0.05; p = 0.35). The relationship between the pre-collection CD 34+ cell concentration in blood and the numbers of CD 34+ cells collected could also be considered as linear with the correlation coefficients in CMNC Spectra Optia R = 0.93, and Cobe Spectra R = 0.78, respectively. In LVL, the median platelet loss was significantly lower in CMNC Spectra Optia (45%) than in Cobe Spectra (57%). Twelve patients were transplanted using PBPC prepared in the standard regimen and 44 using PBPC from LVL. Median time to neutrophil reconstitution in the standard regimen was 11 days for both CMNC Spectra Optia and Cobe Spectra, while platelet reconstitution was 14 days for CMNC and 12 days for Cobe Spectra. The median time to neutrophil and platelets reconstitution in LVL was the same for CMNC Spectra Optia and, corresponding to 11 and 13 days, respectively. Conclusions: The CMNC Spectra Optia protocol is a modern, efficient and safe system that can be used for both Standard and LVL procedures. In well mobilized patients, a sufficient dose of CD 34+ cells for transplantation could be prepared from one Standard or one LVL procedure. No serious adverse reactions have been observed.

• Keywords
• Optia CMNC,
• MeSH
• Antigens, CD34 MeSH
• Transplantation, Autologous MeSH
• Adult MeSH
• Evaluation Studies as Topic MeSH
• Peripheral Blood Stem Cells * MeSH
• Middle Aged MeSH
• Humans MeSH
• Hematopoietic Stem Cell Mobilization MeSH
• Aged MeSH
• Cell Separation methods   MeSH
• Blood Component Removal MeSH
• Peripheral Blood Stem Cell Transplantation * MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH