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Mesenchymal stem/stromal cells (MSCs) show excellent therapeutic potentials in many preclinical studies and clinical trials. However, the clinical application of conventional tissue-derived MSCs faces challenges of limited scalability and high donor variations. To address these challenges, we established a protocol for deriving and characterizing MSCs from human induced pluripotent stem cells (iPSCs) with a theoretically limitless expandability. The iPSC-MSCs show biological properties comparable to or better than early passage bone marrow MSCs and can be scaled up to huge amounts with uniform properties.

Background. Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods. Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results. Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion. Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

Background: The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) of preclinical models including patient-derived xenografts (PDX), organoids (PDOrg) and patient-derived cell cultures (PDC). Extensive clinical annotation and genomic datasets are available for these preclinical models. However, it is unclear if the molecular profiles of the corresponding patient tumors are stably propagated in these models. We have previously demonstrated that PDX models from the NCI PDMR faithfully represent the patient tumors both in terms of genomic stability and tumor heterogeneity. Here, we conduct an in-depth investigation of genomic representation of patient tumors in the PDOrgs and PDCs. Methods: PDOrgs (n=64) and PDCs (n=94) were established from tumor fragments (i.e., initiator specimens) obtained either from patient specimens or from PDX specimens of early passage. For some models (n=19), both PDOrgs and PDCs were generated from the same tumor tissue; in fewer cases (n=4), PDCs were established from organoids derived from patient specimens. Whole Exome Sequencing and RNA-Seq were performed on all PDCs and PDOrgs, and data were compared with patient specimens or early passage PDXs. Results: A majority of the PDOrgs and PDCs have stably inherited the genome of the corresponding patient specimens based on the following observations: (1) >87% of PDOrgs and PDCs maintained similar copy number alteration profiles compared with the initiator specimens of the preclinical model; (2) the variant allele frequency (VAF) of clinically relevant mutations remained consistent between the PDOrgs, PDCs, and the initiator specimens, with none of the PDCs or PDOrgs deviating by >15% VAF; and (3) clinically relevant biomarkers (e.g., MSI, LOH, mutational signatures etc.) are concordant amongst the PDOrgs, PDCs, and the initiator specimens. We observed that the majority of SNVs and indels present in the initiator specimens were also found in the PDOrgs and PDCs, suggesting almost all the tumor heterogeneity was preserved in these preclinical models. Conclusions: This large and histologically diverse set of PDOrgs and PDCs from the NCI PDMR exhibited genomic stability and faithfully represented the tumor heterogeneity observed in corresponding patient specimens. These preclinical models thus represent a valuable resource for researchers interested in pre-clinical drug or other studies. Citation Format: Biswajit Das, Yvonne A. Evrard, Li Chen, Rajesh Patidar, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm-Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Carrie A. Bonomi, Kelly M. Dougherty, Joseph P. Geraghty, Marion V. Gibson, Savanna S. Styers, Abigail J. Walke, Jenna E. Moyer, Anna Wade, Mariah L. Baldwin, Kaitlyn A. Arthur, Kevin J. Plater, Luke Stockwin, Matthew R. Murphy, Michael E. Mullendore, Dianne L. Newton, Melinda G. Hollingshead, Chris A. Karlovich, Paul M. Williams, James H. Doroshow. Patient-derived organoid and cell culture models from the NCI Patient-Derived Models Repository (NCI PDMR) preserve genomic stability and heterogeneity of patient tumor specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3916.

Stem cells can differentiate into various body tissues and organs and thus are considered as promising tools for cell therapy and tissue engineering. Early passage stem cells have high differentiation ability compared to late passage stem cells. Thus, it is important to use early passage stem cells in cell therapy. Here, we investigated whether cell migration could be used to compare young and senescent cells. We used ‘electrotaxis’ where cells under electric treatment move towards the anode or cathode. Without an electric stimulus, stem cells moved randomly. However, under a direct electric current, the cells moved with directionality. Under stimulation with a direct electric current, early passage stem cells moved towards the anode; when the cells became senescent with increasing passages, the percentage of cells migrating to the anode decreased. These results suggest that the behavior of stem cells under the influence of a direct electric current is also related to their passage number. Therefore, electrotaxis migration analysis can be used to distinguish between young cell and senescent cells. Keywords: Stem cell, Directional migration, Electrotaxis, Senescence

This article analyses the sociopolitical interactions that shaped an early colonial union statute that constituted a milestone because of its early passage, draconian nature and wider influence in the British Empire. We analyse the interactions between the Governor on the one hand and local and international actors on the other, to create the first trade union law in Mauritius. Colonial Office officials pressured the Governor to overcome local resistance to legislation, but this opposition and his attitudes shaped the law's content.

The dermal papilla (DP) plays pivotal roles in hair follicle morphogenesis and cycling. However, our understanding of the biology of the canine DP is extremely limited. The aim of this study was to elucidate molecular biological and immunohistochemical characteristics of canine DP cells and determine appropriate conditions for in vitro expansion. Histological investigation revealed that the canine DP expressed biomarkers of human and rodent DP, including alkaline phosphatase (ALP) and versican. When microdissected, canine DP, but not fibroblasts, strongly expressed the DP-related genes for alkaline phosphatase, Wnt inhibitory factor 1 and lymphoid enhancer-binding factor 1, confirming successful isolation. The growth rate of isolated canine DP cells was moderate in conventional culture conditions for rodent and human DP; however, AmnioMAX-C100 complete medium allowed more efficient cultivation. Dermal papilla marker gene expression was maintained in early passage cultured DP cells, but gradually lost after the third passage. Approaches to mimic the in vivo DP environment in culture, such as supplementation of keratinocyte-conditioned medium or use of extracellular matrix-coated dishes, moderately ameliorated loss of DP gene expression in canine DP cells. It is possible that constituent factors in AmnioMAX may influence culture. These findings suggested that further refinements of culture conditions may enable DP cell expansion without impairing intrinsic properties and, importantly, demonstrated that AmnioMAX-cultured early passage canine DP cells partly maintained the biological characteristics of in vivo canine DP cells. This study provides crucial information necessary for further optimization of culture conditions of canine DP. Resume La papille dermique (DP) joue un role essentiel dans la morphogenese et le cycle du follicule pileux. Toutefois, notre comprehension de la biologie de la DP canine est extremement limitee. L’objectif de cette etude etait de determiner les caracteristiques des cellules de DP canine ainsi que les conditions appropriees pour leur expansion in vitro. L’analyse histologique a revele que la DP canine exprimait les biomarqueurs de DP humaine et de rongeurs, regroupant la phosphatase alcaline (ALP) et Versican. Apres dissection, la DP canine mais pas les fibroblastes, exprimait fortement les genes lies a l’ALP de DP, WIF1(Wnt inhibitory factor 1) et LEF1 (Lymphoid enhancer-binding factor 1), confirmant le succes de l’isolement. Le taux de croissance des cellules de DP canine isolees etait modere sous des conditions de culture conventionnelle pour DP d’humains et de rongeurs. Cependant le milieu complet AmnioMAX-C100 a permis une culture plus efficace. L’expression du gene marqueur etait maintenue dans les premiers passages de cultures de cellules de DP mais etait progressivement perdue apres le troisieme passage. Les essais pour mimer l’environnement in vivo de DP en culture, comme la suplementation avec milieu de culture conditionnes pour keratinocytes ou l’utilisation de plaques revetues de matrices extracellulaires, ont permis une amelioration moderee de la perte de l’expression de gene de DP par les cellules canines de DP. Il est possible que les facteurs constituants de AmnioMAX influencent la culture. Ces resultats suggerent que des affinements supplementaires des conditions de culture pourraient permettre l’expansion des cellules de DP sans compromettre leurs proprietes intrinseques et surtout, que les premiers passages de culture de cellules de DP canines sur AmnioMAX conservaient partiellement les caracteristiques biologiques des cellules de DP canines in vivo. Cette etude fourni une information cruciale necessaire pour l’optimisation des conditions de futures cultures de DP canines. Resumen La papilla dermica (DP) juega un papel esencial en la morfogenesis y el ciclo del foliculo piloso. Sin embargo, nuestro entendimiento de la biologia del la DP canina es muy limitado. El proposito de este estudio fue elucidar las caracteristicas moleculares, biologicas e inmunohistoquimicas de las celulas de la DP y determinar la condiciones mas apropiadas para la expansion in vitro. La investigacion histologica revelo que la DP canina expresaba biomarcadores de la DP de humanos y de ratones, incluida la fosfatasa alcalina (ALP) y el versican. Cuando se utilizo microdiseccion, las celulas de la DP canina, pero no los fibroblastos, expresaron intensamente genes relacionadas con la DP como ALP, factor de inhibicion Wnt-1 (WIF-1) y el factor de estimulacion de union linfoide-1 (LEF-1), confirmando un aislamiento exitoso. El ritmo de crecimiento de las celulas aisladas de la DP canina fue moderado bajo condiciones convencionales de cultivo para celulas de la DP de ratones y de humanos, sin embargo, medio completo AmnioMAX-C100 permitio un cultivo mas eficiente. Se mantuvo la expresion de los marcadores de la DP en las celulas de los pases tempranos en cultivo, pero gradualmente se perdieron tras el tercer pase. Intentos de reproducir la condiciones del microambiente de la DP in vivo en cultivo, tal como el suplemento con medio condicionado para queratinocitos o el uso de placas cubiertas con matriz extracellular, ligeramente mejoraron la perdida de los genes de la DP en la celulas. Es posible que factores constitutivos del medio AmnioMAX influyan el cultivo. Estos hallazgos sugieren que es necesario mejorar las condiciones de cultivo para permitir la expansion de las celulas de la DP sin alterar las propiedades intrinsecas, y, lo que es mas importante, demostro que pases iniciales de celulas DP en cultivo con medo AmnioMAX mantenian parcialmente las caracteristicas de las celulas de la DP in vivo. Este estudio aporta informacion crucial necesaria para posterior optimizacion del las condiciones de cultivo de celulas de la DP canina. Zusammenfassung Die dermale Papille (DP) spielt eine entscheidende Rolle bei der Morphogenese des Haarfollikels und des Haarzyklus. Nichtsdestotrotz ist unsere Kenntnis der Biologie der caninen DP stark limitiert. Es war das Ziel dieser Studie, die molekularbiologischen und immunhistochemischen Merkmale der caninen DP Zellen zu beleuchten und geeignete Bedingungen fur ihre Entwicklung in vitro zu bestimmen. Die histologische Untersuchung zeigte die Expression von Biomarkern der Papilla des Menschen und der Nager, ebenso der alkalischen Phosphatase (ALP) und von Versican. Nach der Mikrodissektion bestand eine starke Expression der caninen DP (nicht aber der Fibroblasten) von DP-verwandten Genen ALP, Wnt Inhibitory Factor 1 (WIF1) und Lymphoid enhancer-binding factor 1 (LEF1), was eine erfolgreiche Isolierung bestatigte. Die Wachstumsrate von isolierten caninen DP-Zellen war bei konventionellen Kulturbedingungen fur die DP des Menschen und der Nager masig, AmnioMAX-C100 Komplettmedium erlaubte jedoch eine effizientere Kultivierung. Die Expression von DP Markergenen war in DP-Zellen der Fruhkultur erhalten, ging aber nach der dritten Passage graduell verloren. Methoden, die Umgebung von DP in vivo in der Kultur nachzuahmen, wie etwa die Supplementierung von Keratinozyten-konditioniertem Medium oder der Verwendung von extrazellularen mit Matrix ausgekleideten Gefasen, verringerte den Verlust der DP Genexpression in den caninen DP Zellen nur masig. Es besteht die Moglichkeit, dass Bestandteile von AmnioMAX die Kultur beeinflussen konnten. Diese Ergebnisse geben Hinweise darauf, dass eine weitere Vervollkommnung der Kulturbedingungen das Wachstum von DP Zellen ermoglichen konnte ohne ihre intrinsischen Eigenschaften zu beeintrachtigen und es wurde etwas sehr Wesentliches gezeigt, namlich dass caninen DP Zellen einer Fruhkultur in AmnioMAX teilweise die biologischen Charakteristika der caninen DP Zellen in vivo erhalten geblieben sind. Diese Studie liefert wichtige Informationen, die fur eine zukunftige Optimisierung der Kulturbedingungen fur canine DP Zellen essentiell sind.

12023Background: Patient-derived xenografts (PDXs) are a powerful tool for cancer translational research. However, it is unclear if early passage PDXs faithfully recapitulate the molecular profiles...

Aim: This study aimed to assess genetic changes during long term culturing of two malignant mesothelioma (MM) cell lines (MM04 and MM05). Results and Discussion: Spindle shaped cells were observed in both passages for both cell lines. TEM also confirmed the same morphology in early and late passages in MM05. Doubling time experiments confirmed faster growth of late passage versus early passage cultures. ((MM05 (P9) 45.49 hours vs. (P47) 43.28 hours). Doubling time experiments for MM04 late passage is underway (The result will be presented at the conference). T Test (p < 0.001) on gene expression profile displayed down regulation of 8 genes (TMEM165, GREB1, HS566426, PRUNE, SLC10A7, C4ORF38, TBN, USP53) and up regulation of 6 genes (TMEM2, CDKAL1, TIAM2, RPL23A, STAT3, UPK1B) were found to be in late passages (figure). The genetic imbalances occurring in early to late culture steps reflected the tendency of MM cells toward genetic changes during long term culturing. © 2010 The Author(s) Journal Compilation © 2010 Asian Pacific Society of Respirology

Human embryonic stem cell (hESC) growth is dependent on various factors released by feeder cells. Some of them have already been elucidated, although much research is still needed to understand the biology of stem cell maintenance in culture. Traditionally, primary mouse embryonic fibroblasts (PMEFs) have been used as feeder layers, and both murine and human fibroblast cell lines have been shown to support pluripotency and self-renewal of hESC. Here we report the derivation of three new mouse embryonic fibroblast cell lines, MEFLU-M, MEFLU-T, and MEFLU-TB, with different properties regarding growth and support for undifferentiated hESCs. MEFLU-TB is able to support continuous growth of the newly derived Man-1, as well as H1, HUES-1, HUES-7, HUES-8, and HUES-9 human embryonic stem cell lines. After more than 50 passages and doublings, MEFLU-TB feeders compare to early passage primary mouse embryonic fibroblasts in their ability to support undifferentiated hESC growth. Our results contradict a previous paradigm that PMEFs tend to lose their capacity to support proliferation of hESCs with increasing passages, and show that the MEFLU-TB mouse embryonic fibroblast cell line and its conditioned medium have the potential to support the maintenance of hESC lines. Also, our results clearly show that spontaneous immortalization of primary fibroblasts can be achieved in culture without any chemical addition or genetic modification.

Severe burns can be very traumatic for the patient, and while burns caused by industrial or domestic accidents are common, there are also increasing numbers of burns associated with terrorism. A novel technique to assist in the healing process is to spray skin cells, keratinocytes, that are cultured from the patient's own tissue, directly onto the burn site. This process involves taking some undamaged skin from the patient, allowing the skin cells to proliferate rapidly in the laboratory over a period of 5-10 days, harvesting and separating the cells and then spraying them onto the burn. This paper deals with keratinocytes that have been cultured in vitro for a short period of time (early passage cultured cells). The spraying process has yet to be optimised with respect to the seeding density required for fastest re-epithelisation and thus there is a need for this process to be modelled. In this paper, we review some of the skin biology and develop a mathematical model of the growth patterns of cell colonies after they have been applied using a aerosolised technique. The model allows us to predict coverage over time and can be used as a decision support tool for clinicians.

This study reports on a pediatric case of hemangiopericytoma (HPC) showing trisomy 15 as a sole anomaly. Trisomy 15 was observed in a total of 11 cells harvested at a very early passage from two different in-situ cultures. Trisomy 15, as a sole anomaly, has been described in hematologic disorders such as myelodysplastic syndromes but, to our knowledge, has never been documented in solid tumors. This is the first report of HPC with trisomy 15.

We found that exposure to room temperature (RT/21°C) causes apoptosis in HL-60 cells. Here we characterized RT-induced apoptosis in HL-60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase-1 inhibitor (YVAD-CHO) effectively blocked apoptosis, whereas the caspase-3 inhibitor (DEVD-CHO) did not. About 20% of newly obtained early passage HL-60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL-60 which responds to RT with apoptosis became dominant during passaging.

The culture medium supplemented with carp serum and fetal bovine serum (FBS) promoted cell growth significantly and induced morphological change of goldfish fin cells in early passage as compared to the medium containing FBS alone. However, these effects were not observed in RBCF-1, a cell line established from the goldfish fin. The sensitivity of the cells in early passage to carp serum suggests the following possibilities: (1) cells in early passage retain the ability to respond to growth-promoting factors specifically included in carp serum; and (2) this ability is lost during the process of long-term culture and/or long-term culture in FBS eliminates cell groups showing high dependency of cell growth on carp serum.

Two things happen every time an employee retires, is made redundant or leaves to join another company. The experience acquired at the company’s considerable expense literally walks out of the door and the company has to replace the individual, often from outside. For organizations, job change, which has always been continuous, has been accelerating over the past decade as individuals switch their employer every six years. Even though most professionally managed companies run induction courses, most new managers will readily admit that the components that most inhibit their early passage to full productivity relate to understanding and accommodating their new employer’s individual corporate culture, management and communication styles, and the detail of recent events. Because of the difficulties of imparting such information, companies generally leave individuals to assimilate these intangibles as best they can, usually by osmosis. The evidence suggests that it can take 12 months ‐ and often much longer ‐ to become fully productive. Lack of continuity has an insidious effect on productivity and competitiveness. Outlines a cost‐effective solution to high job mobility.

The performance of two proteomic sample preparation methods, "pseudoshotgun" (PSG) and filter-aided sample preparation (FASP) were compared in terms of the number of identified proteins, representation of cellular component GO (gene ontology) categories in the obtained list of proteins, and the efficiency of both methods in the proteomic analysis of a very low number of cells. Both methods were combined to obtain a proteomic profile of a short-term culture (passage 3) of melanoma cells, established in our laboratory from a human metastatic melanoma lesion. The data revealed that with FASP, usually more proteins are identified than with PSG when analyzing a higher number of cells (≥ 5000/injection), whereas PSG is favorable when analyzing only a very small amount of cells (250-500/injection). PSG and FASP, however, are complementary techniques, as combining both methods further increases the number of identified proteins. Moreover, we show that it is feasible to identify a substantial number of proteins from only 250 cells/injection that is equivalent to 60 ng of protein.

Biobanking became a necessity for translating genetic discoveries into clinical practice. Approaches to personalized medicine require a new model system for functional and pharmacogenomic studies of a variety of accumulating genetic variations, as well as new research environments such as biobankomics. Human lymphoblastoid cell lines (LCLs) will provide a valuable tool to meet such new demands in the biobankomics era. The National Biobank of Korea (NBK), which is leading the Korea Biobank Project, has a large collection of LCLs derived mostly from population-based cohort samples. Using a special long-term subculture collection of NBK LCLs, biological characteristics of early passage LCLs and terminally immortalized LCLs have been investigated to promote the utilization of LCLs and provide well quality-controlled LCLs for genetic and pharmacogenomic studies. As LCLs have been successfully phenotyped for cytotoxicity in response to various stimulators, including chemotherapeutic agents, environmental chemicals and irradiation, the utility of LCLs will increase in the future. Here, we discuss current and future applications of NBK LCLs for the biobankomics era.

We have established two glial fibrillary acidic protein (GFAP)-positive human glioma cell lines and have analyzed their stability of GFAP expression through successive passages using the immunohistochemistry and Northern blot hybridization. In one cell line (Case 1), the cultured cells showed stable expression of GFAP protein and mRNA from early to later passage levels. Stability of GFAP expression was also shown in all of 8 clones isolated from this cell line. In the other cell line (Case 2), GFAP expression was shown to be less stable. At early passage, nearly 100% of the cultured cells displayed intense GFAP immunoreactivity

Human multipotential stromal cells (hMSCs) are easily isolated from bone marrow and can be expanded by up to 200-fold in culture. Cultures of hMSCs are heterogeneous mixtures of stem/progenitor cells and more mature cell types. The proportion of each cell type in a given culture depends on how the cells are maintained. To maintain their stem cell-like qualities, hMSCs should be plated at low seeding densities (60-150 cells/cm2), lifted when between 60% and 80% confluent and should not be expanded beyond 4-5 passages. Thus, it is useful to establish a frozen bank of early passage cells. hMSCs store well in vapor phase liquid nitrogen (LN2) and are easily recovered for further expansion. This chapter describes one method of establishing a bank of early passage hMSCs using a seed lot system and the subsequent recovery of hMSCs from frozen stocks. The recovered cells can then be harvested and used for analyses of identification, functionality, in vitro and/or in vivo experimentation, or further expanded.

The cellular response to mineral trioxide aggregate (MTA) is important for the repair and regeneration of periradicular tissues. The purpose of this study was to analyze the response of human alveolar bone cells to MTA. A human alveolar bone chip was obtained from an oral surgical procedure and explant cultures harvested after 3 to 4 weeks of outgrowth in α-minimum essential medium supplemented with fetal calf serum. Cells in early passage were seeded onto preset ProRoot (gray) MTA, tooth-colored (white) MTA, and MTA prepared with local anesthetic solution. Scanning electron microscopy showed cells were attached and spread out onto MTA within 24 hours, and proliferated to form a matrix-like layer within 7 days. Cell attachment and cell-surface interactions with the gray and white MTA, and with the MTA prepared with local anesthetic were comparably propagated for 14 days. The surgically derived human alveolar bone cells provided a clinically relevant model that demonstrated the capacity of both ProRoot and tooth-colored MTA to support cell attachment, proliferation, and matrix formation.

We have previously reported that tranilast, an anti-allergic agent, specifically suppresses collagen synthesis in normal skin fibroblasts and to a greater extent in keloid fibroblasts. We found in this study that the specific suppression of collagen synthesis by tranilast was limited to the fibroblasts with a high passage number (passage 8–10). In normal skin fibroblasts with a low passage number (passage 1–2), tranilast exerted no significant effect on collagen synthesis. This was also observed with scleroderma and keloid fibroblasts. This result suggests that inhibition of collagen by tranilast will be dependent on in vitro cellular aging and that serial cell passages result in the loss of the cell phenotype resistant to tranilast effect.

Cultured human umbilical vein endothelial cells (HUVECs) are a valuable model for investigation of endothelial functions, but they enter senescence at low passage. Transfection of early passage HUVECs with the early genes of SV40 greatly extends the replicative potential of these cells, but eventually results in marked changes in growth, morphology, and biochemistry. Here we report a modified approach that appears to have overcome the problem of late passage decline after transfection. Plasmid pX-8 containing the SV40 early genes was transfected into passage four HUVECs. At passage five, these transfectants were cloned by limiting dilution and selected on the basis of both morphological and biochemical resemblance to their untransfected counterparts. Two clones that expressed factor VIII and in which the basal and the tumor necrosis factor-α inducible levels of interleukin 6 and endothelial adhesion molecules were normal were chosen. Vimentin and fibronectin distribution in these clones resembled untransfected cells. At passage 25, growth pattern changes were becoming evident, but recloning these late passage clones recovered numerous subclones of normal, cobblestone appearance. Two of these were further characterized and found to resemble their original parental clone by all of the biochemical criteria listed above. These subclones appeared to transform more rapidly than the parental clone, but repeated subcloning again rescued clones with normal morphologies and normal biochemical characteristics. We conclude that periodic recloning may indefinitely perpetuate lines that are useful equivalents of their original counterparts.Key words: SV40, endothelial, immortalized, adhesion molecules.

The growth patterns of established cell lines from bladder transitional cell carcinoma (TCC) were compared with early passage cell lines. The growth of established cell line 5637 was uninhibited in both serum free (basal) and serum containing media. The early passage line (DR) grew only in serum containing medium. This confirms the unreliability of results from biological studies on established (continuous) cell lines.

Due to the lack of an appropriate cell source, external ear reconstruction based on tissue engineering techniques has not yet been translated to clinical applications. It has been reported that the chondrocytes from microtic cartilage can be used for cartilage regeneration. However, in our prior study, we found the passaged microtic chondrocytes became less proliferative and decreased expression of cartilage-specific markers, which would lead to the limited quantity and quality of cells from microtic cartilage to engineer an entire ear. To make full use of these cells, it is important to know whether they can provide the chondrogenic niche to promote the chondrogenesis of BMSCs. In this study, through the co-transplantation of BMSCs with microtic chondrocytes subcutaneously into nude mice, we showed that microtic chondrocytes have the potential to promote ectopic chondrogenesis of BMSCs. These results indicate that the early passage of chondrocytes from the microtic cartilage might have the same potential to promote chondrogenesis of BMSCs as normal chondrocytes. The co-culture of microtic chondrocytes with BMSCs potentially offers a practical approach to autologous ear regeneration.

The susceptibility of early-passage feline brain cells and Crandell feline kidney (CRFK) cells to infection with three isolates of feline immunodeficiency virus (FIV) was investigated. The Petaluma strain of FIV could well infect both the feline brain cells and CRFK cells. The KYO-1 strain could well infect the feline brain cells but the replication in CRFK cells was demonstrated only by coculturing fresh feline T-lymphoblastoid cells with the infected cells. On the other hand, the TM1 strain could infect the feline brain cells but not CRFK cells. Moreover, the replicative ability of the TM1 strain in the feline brain cells was much less than the KYO-1 and Petaluma strains. These results indicate that biological differences can be detected among the FIV isolates.

In October 2003, the National Institutes of Health (NIH) established three extramural “Exploratory Centers for Human Embryonic Stem Cell Research.” Our Center’s experience in acquiring and manipulating NIH-approved human embryonic stem (hES) cell lines may be useful for other institutions interested in pursuing NIH-funded hES cell research. We acquired 14 of the 22 NIH-approved cell lines. Modifications to the proposed Material Transfer Agreements (MTAs) with hES cell suppliers were sought to improve accessibility to these hES cell lines by local researchers. Lines were characterized for survival following cryopreservation and for their ability to adapt to a uniform set of culture conditions while maintaining a normal karyotype. Each of the 4 suppliers contacted eventually agreed to terms that improved access to their hES cell lines. Eleven hES cell lines were received frozen, and in 10 cases very few cells survived cryopreservation. Ten hES cell lines were successfully converted to simplified culture conditions that enhanced their ability to be maintained and expanded in culture. One hES cell line had an unstable karyotype at an early passage. Current MTA provisions continue to present significant obstacles to NIH-funded hES cell research. Many hES cell lines can be maintained using culture conditions less onerous than those recommended by their suppliers.

A chromosome polymorphism was detected between two early passage euploid Chinese hamster cell strains when a fluorescence shift of the small metacentric No. 9 chromosome was resolved by flow cytometry. The characteristics of the polymorphism were studied using cultures established from ear clippings taken from 16 additional hamsters from our breeding colony. Additional variants of chromosome 9 were detected using flow cytometry, and a subset of these variants were analyzed by G- and C-banding. An increase of fluorescence recorded by flow cytometry correlated with an increase of centromeric heterochromatin. Autosomal normalization of the flow karyotype from 18 different animals indicated three distinct peak positions for chromosome 9. The results indicate that a discrete block of constitutive heterochromatin may be present in one or two extra copies within the small inbred colony of hamsters studied. To determine the inheritance patterns, hamsters with known polymorphic No. 9 chromosomes were bred. The flow karyotypes derived from the offspring of these matings provide strong evidence that chromosomal polymorphisms are inherited in Mendelian fashion.

Previous studies have shown that when different naturally occurring cells that display a measurable G1 during logarithmic growth (G1 + cells) were hybridized, the resulting hybrids often lacked G1 (G1 − cells). This has been termed complementation. Four established G1 + lines of Chinese hamster were found to define three G1 + complementation groups. Three of these lines have been hybridized with early and late passage fibroblast-like cells from the lung and liver of a Chinese hamster newborn. The complementation tests indicate: 1. 1. that the basis for the expression of G1 of the ‘lung’ cultures is different than that of the ‘liver’ cells 2. 2. that late passage (transformed) cells retain the same basis for G1 as their ‘ancestral’ early passage cells. These results show that presumably different cells can express G1 for different reasons, which in turn might be related to cellular differentiation.

The quantity and type of microsomal cytochrome P-450 was determined in early passage cultured fibroblasts from six mammalian species of widely differing life spans. The proportion of cytochrome P-450 and P448 varied considerably among species, with the shorter-lived species having a preponderance of cytochrome P-448. There was a good inverse correlation between potential species life span and the specific cytochrome P-448 content of cultured fibroblasts.

DNA replication of cultured fibroblasts of early passage derived from Werner's syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werner's syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werner's syndrome cells and normal cells.

Human cells in early passage cultures of benign oral tumors, normal oral tissues, normal lung tissues and, especially, in long-term established oral carcinoma cultures were highly susceptible to infection by human adenovirus types 5, 21, and 31. In contrast, replication of each adenovirus type was markedly limited in inoculated cells of newly-established oral squamous cell carcinoma cultures.

We have investigated X-ray survival parameters and repair of potentially lethal damage ( PLDR ) in ten early passage squamous cell carcinoma cell lines derived from patients who were biopsied before initiation of radiotherapy or after radiation therapy failure. Radiosensitivity (D0) ranged from 1.07 to 1.93 (Gy), extrapolation numbers (-n) from 1.17 to 2.14 and PLD recovery at 24 h from 1.4 to 20.3. Despite significant differences in these parameters amongst the cell lines, a firm correlation between radiocurability and any individual radiobiological parameter could not be established. Our data suggest that the mechanisms associated with radioresistance are complex and that any single radiobiological parameter may not predict clinical success or failure.

To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal an environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 microgram) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating particles and procedures provided adequate safeguards for personnel and the environment.