Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

IPPRAS Cryobank for the Conservation of Orthodox Seeds of Rare, Endangered, Medicinal, and Ornamental Plant Species-Current Research.

Plant cryobanks play a significant role in modern science and breeding. They contribute to the recovery of lost species, the emergence of new plant varieties, and help preserve and explore the diversity of the plant world. The IPPRAS Cryobank collection is constantly supplemented with new samples, while, at the same time, the stored samples are being monitored. In order to test seed germination, seeds of Allium and Veratrum species were thawed. Rare Allium species seeds, such as A. nutans, A. schoenoprasum, and A. victorialis were stored in liquid nitrogen for 17, 19, and 30 years, respectively. Long-term cryopreservation decreased germination rates for A. nutans from 96.55 to 50.00%, for A. schoenoprasum from 72.00 to 62.75%, and for A. victorialis from 90.00 to 83.05%. Seeds of a rare medicinal species, Veratrum lobelianum, were stored in liquid nitrogen for 18 years; the seed germination rate during this storage period has been significantly decreased from 75.00 to 14.81%. V. nigrum seeds were also collected and frozen in liquid nitrogen for 3 days. Short-term cryopreservation did not result in a statistically significant change in germination rates (from 79.71 to 82.69%). The seeds of an endangered ornamental species, Cypripedium calceolus, were collected and kept frozen for 3 days. After cryopreservation, the seeds were planted on three different media, as follows: ½ MS, MS with 10% coconut milk, and BM1. On ½ MS medium, 24.98% seeds formed protocorms, while on MS medium with 10% coconut milk, this number was 10.02%, and on BM1 medium, it was 15.02%, respectively; however, after 2.5 months, all of the protocorms died. Thus, it appears that the existing protocol for seed cryopreservation of C. calceolus needs further improvement. The size, weight, and free water content (WC) of six previously cryopreserved Stipa species and three Allium species were measured. For all the Allium and Stipa species studied, we found no correlation between seed size, WC, and cryotolerance. We also found no correlation between the life form, which reflects the water requirement of the species, and cryotolerance.

A SIMPLE, QUICK, AND ECONOMICAL METHOD FOR IN VITRO CULTIVATION OF ECHINOCOCCUS MULTILOCULARIS METACESTODE AND GENERATION OF PRIMARY CELLS.

Alveolar echinococcosis is considered to be one of the most potentially lethal parasitic zoonotic diseases. However, the molecular mechanisms by which Echinococcus multilocularis interacts with hosts are poorly understood, hindering the prevention and treatment of this disease. Due to the great advantages of cell culture systems for molecular research, numerous attempts have been made to establish primary cell cultures for E. multilocularis. In this study we developed a simple, rapid, and economical method that allows E. multilocularis metacestode tissue blocks to generate daughter vesicles without the continuous presence of host feeder cells in a regular medium. We performed anaerobic, hypoxic (1% O2), normoxic, and semi-anaerobic (in sealed tubes) cultures and found that E. multilocularis metacestode tissues can produce daughter vesicles only in the sealed tubes after 4 wk of incubation. The daughter vesicles cultivated in this system were remarkably enlarged under anaerobic conditions after 8 days of culture, whereas vesicles cultured under hypoxic (1% O2) and normoxic conditions showed only a mild increase in volume. Our in vitro cultivated vesicles showed strong viability and could be used to test antiparasitic drugs, isolate primary cells, and infect animals.

Strategies, Achievements, and Potential Challenges of Plant and Microbial Chassis in the Biosynthesis of Plant Secondary Metabolites.

Diverse secondary metabolites in plants, with their rich biological activities, have long been important sources for human medicine, food additives, pesticides, etc. However, the large-scale cultivation of host plants consumes land resources and is susceptible to pest and disease problems. Additionally, the multi-step and demanding nature of chemical synthesis adds to production costs, limiting their widespread application. In vitro cultivation and the metabolic engineering of plants have significantly enhanced the synthesis of secondary metabolites with successful industrial production cases. As synthetic biology advances, more research is focusing on heterologous synthesis using microorganisms. This review provides a comprehensive comparison between these two chassis, evaluating their performance in the synthesis of various types of secondary metabolites from the perspectives of yield and strategies. It also discusses the challenges they face and offers insights into future efforts and directions.

Challenges and opportunities in cell expansion for cultivated meat.

The cultivation of meat using in vitro grown animal stem cells offers a promising solution to pressing global concerns around climate change, ethical considerations, and public health. However, cultivated meat introduces an unprecedented necessity: the generation of mass scales of cellular biomaterial, achieved by fostering cell proliferation within bioreactors. Existing methods for in vitro cell proliferation encounter substantial challenges in terms of both scalability and economic viability. Within this perspective, we discuss the current landscape of cell proliferation optimization, focusing on approaches pertinent to cellular agriculture. We examine the mechanisms governing proliferation rates, while also addressing intrinsic and conditional rate limitations. Furthermore, we expound upon prospective strategies that could lead to a significant enhancement of the overall scalability and cost-efficiency of the cell proliferation phase within the cultivated meat production process. By exploring knowledge from basic cell cycle studies, pathological contexts and tissue engineering, we may identify innovative solutions toward optimizing cell expansion.

Use of image analysis to assess radial growth of Passalora arachidicola and Nothopassalora personata on solid media.

Despite significant research on early and late leaf spot diseases of peanut, in vitro study of the respective causal agents, Passalora arachidicola and Nothopassalora personata, has been limited due to cultural challenges that make growth of these fungi difficult to quantify with traditional methods. Studies were conducted to evaluate the practicality of image analysis to assess radial growth and tissue volume by correlating these assessments to dry mass. Image analysis was also used to estimate radial growth rates for these fungi over time. Tissue area and volume were significantly correlated to dry mass for P. arachidicola in two separate experiments, and for N. personata when medium had been removed from tissues prior to dry mass assessments. Tissue area densities were the same for P. arachidicola and Pseudocercospora smilacicola, evaluated as a nonstromatal cercosporoid comparison, whereas tissue volume densities were greater for P. archidicola and N. personata than P. smilacicola. A quadratic relationship was observed between radial growth and incubation time for all isolates evaluated. Growth rates of P. arachidicola isolates were 2 to 4 times faster than N. personata during the first week of incubation and slowed over time. Growth rates of NP18R, a phenotype variant of N. personata, increased after neighboring colonies met and was nearly 2.5 times faster than the fastest rates observed for P. arachidicola. These experiments demonstrate that when fungal tissues are observable, image analysis is a useful assessment tool for P. arachidicola and N. personata. Care should be taken to monitor fungal phenotypic changes in these species because phenotype degeneration can affect growth rates.

Cultivation of monoxenous trypanosomatids: A minireview.

Trypanosomatids are obligatory parasites, some of which are responsible for important human and animal diseases, but the vast majority of trypanosomatids are restricted to invertebrate hosts. Isolation and in vitro cultivation of trypanosomatids from insect hosts enable their description, characterization, and subsequently genetic and genomic studies. However, exact nutritional requirements are still unknown for most trypanosomatids and thus very few defined media are available. This mini review provides information about the role of different ingredients, recommendations and advice on essential supplements and important physicochemical parameters of culture media with the aim of facilitating first attempts to cultivate insect-infesting trypanosomatids, with a focus on monoxenous trypanosomatids.

Elucidating the impact of in vitro cultivation on Nicotiana tabacum metabolism through combined in silico modeling and multiomics analysis.

The systematical characterization and understanding of the metabolic behaviors are the basis of the efficient plant metabolic engineering and synthetic biology. Genome-scale metabolic networks (GSMNs) are indispensable tools for the comprehensive characterization of overall metabolic profile. Here we first constructed a GSMN of tobacco, which is one of the most widely used plant chassis, and then combined the tobacco GSMN and multiomics analysis to systematically elucidate the impact of in-vitro cultivation on the tobacco metabolic network. In-vitro cultivation is a widely used technique for plant cultivation, not only in the field of basic research but also for the rapid propagation of valuable horticultural and pharmaceutical plants. However, the systemic effects of in-vitro cultivation on overall plant metabolism could easily be overlooked and are still poorly understood. We found that in-vitro tobacco showed slower growth, less biomass and suppressed photosynthesis than soil-grown tobacco. Many changes of metabolites and metabolic pathways between in-vitro and soil-grown tobacco plants were identified, which notably revealed a significant increase of the amino acids content under in-vitro condition. The in silico investigation showed that in-vitro tobacco downregulated photosynthesis and primary carbon metabolism, while significantly upregulated the GS/GOGAT cycle, as well as producing more energy and less NADH/NADPH to acclimate in-vitro growth demands. Altogether, the combination of experimental and in silico analyses offers an unprecedented view of tobacco metabolism, with valuable insights into the impact of in-vitro cultivation, enabling more efficient utilization of in-vitro techniques for plant propagation and metabolic engineering.

Cultivation of Asexual Intraerythrocytic Stages of Plasmodium falciparum.

Successfully developed in 1976, the continuous in vitro culture of Plasmodium falciparum has many applications in the field of malaria research. It has become an important experimental model that directly uses a human pathogen responsible for a high prevalence of morbidity and mortality in many parts of the world and is a major source of biological material for immunological, biochemical, molecular, and pharmacological studies. Until present, the basic techniques described by Trager and Jensen and Haynes et al. remain unchanged in many malaria research laboratories. Nonetheless, different factors, including culture media, buffers, serum substitutes and supplements, sources of erythrocytes, and conditions of incubation (especially oxygen concentration), have been modified by different investigators to adapt the original technique in their laboratories or enhance the in vitro growth of the parasites. The possible effects and benefits of these modifications for the continuous cultivation of asexual intraerythrocytic stages of P. falciparum, as well as future challenges in developing a serum-free cultivation system and axenic cultures, are discussed.

Investigation of the Copper Requirements of the Metallophyte Liverworts Cephaloziella nicholsonii Douin and C. massalongoi (Spruce) Müll.Frib.

Former mine sites can provide habitat for many rare specialised bryophyte species that have adapted to metal-rich soil conditions that are toxic to most other plant species. Some of the bryophyte species found in this habitat are facultative metallophytes, and others are regarded as strict metallophytes, the so-called 'copper mosses'. It is a general assumption in the literature that Cephaloziella nicholsonii and C. massalongoi, both categorised as Endangered in the IUCN Red List for Europe, are also strict metallophytes and obligate copper bryophytes. This in vitro experiment investigated the growth and gemma production of these two species from different sites in Ireland and Britain on treatment plates of 0 ppm, 3 ppm, 6 ppm, 12 ppm, 24 ppm, 48 ppm and 96 ppm copper. Results show that elevated copper is not an obligate requirement for optimum growth. Differences in response to the copper treatment levels among populations evident within both species could possibly be due to ecotypic variation. A case is also made for the taxonomic revision of the Cephaloziella genus. Implications for the species' conservation are discussed.

Chimaphila umbellata; a biotechnological perspective on the coming-of-age prince's pine.

Chimaphila umbellata has been studied for almost two centuries now, with the first paper exploring the phytochemistry of the plant published in 1860. Almost all contemporary studies focus on the biotechnological advances of C. umbellata including its utilization as a natural alternative in the cosmetic, food, biofuel, and healthcare industry, with a special focus on its therapeutic uses. This literature review critically investigates the significance and applications of secondary metabolites extracted from the plant and presses on the biotechnological approaches to improve its utilization. C. umbellata is home to many industrially and medicinally important phytochemicals, the majority of which belong to phenolics, sterols, and triterpenoids. Other important compounds include 5-hydroxymethylfurfural, isohomoarbutin, and methyl salicylate (the only essential oil of the plant). Chimaphilin is the characteristic phytochemical of the plant. This review focuses on the phytochemistry of C. umbellata and digs into their chemical structures and attributes. It further discusses the challenges of working with C. umbellata including its alarming conservation status, problems with in-vitro cultivation, and research and development issues. This review concludes with recommendations based on biotechnology, bioinformatics, and their crucial interface.

Characterization of the physico-chemical properties of the natural habitat and in vitro culture effects on the biochemistry, proliferation and morphology of Lemna minuta.

In this study, the ecological conditions of the natural habitat of Lemna minuta Kunth in Morocco were investigated, and the impact of five synthetic growth media (Murashige-Skoog (MS), Schenk-Hildebrand (SH), Hoagland medium (HM), 10X Algal Assay Procedure (AAP), and Swedish Standard Institute medium (SIS)) on the morphophysiological and biochemical parameters was analysed. The morphophysiological parameters included root length, frond surface area, and fresh weight, while the biochemical parameters included photosynthetic pigments, carbohydrates, and protein content. The study was conducted in vitro in two phases: an uncontrolled aeration system (Phase I) and a controlled aeration system (Phase II).The results showed that the pH, conductivity, salinity, and ammonium levels in the natural habitat were within the optimal range for duckweed growth. The measured orthophosphate concentrations were higher compared to previous observations, while the recorded chemical oxygen demand values were low. The study also revealed a significant effect of the culture medium composition on the morphophysiological and biochemical parameters of the duckweed. The fresh weight biomass, relative growth rate in fronds, relative growth rate in surface area, root length, protein content, carbohydrates, chlorophyll (a), chlorophyll (b), total chlorophyll, carotenoids, and the chlorophyll (a/b) ratio were all affected by the culture medium.The most accurate regression models described the growth index GI(F) based on time and in vitro culture conditions in both phases. In Phase I, the best models for MS, SIS, AAP, and SH media were linear, weighted quadratic, cubic, and weighted cubic, respectively. In Phase II, the best models for all growth media were linear. The time coefficients (in days) for Phase II were 0.321, 0.547, 1.232, 1.470, and 0.306 for AAP, HM, MS, SH, and SIS, respectively.Comparing the morphophysiological and biochemical parameters of fronds from different media and analysing the regression model results showed that the SH and MS media were the best among the tested media for the in vitro culture of L. minuta in controlled aeration conditions. However, further research is needed to develop new synthetic media that best promote the growth and maintenance of this duckweed in long-term culture.

Acid scarification as a potent treatment for an in vitro germination of mature endozoochorous Vanilla planifolia seeds.

BACKGROUND: Vanilla planifolia is the most widely cultivated species of vanilla with high economic importance. However, seed germination under artificial conditions is difficult and yields low germination percentages. The seeds are adapted to endozoochorous dispersal, and we therefore tried to simulate the conditions in the digestive tract by acid scarification of seeds. RESULTS: Immature seeds lacking dormancy, used as a control, showed the highest germination percentage. Among the treatments tested for mature seeds, the hydrochloric acid treatments were significantly the best in breaking dormancy and inducing germination, irrespective of the acid concentration and the presence of pepsin. Conventional treatment with a hypochlorite solution induced much lower germination percentage. Sulphuric acid at concentration 50% was too strong and caused damage to the seeds. Important factor is also high cultivation temperature 30 °C as there was nearly no germination at 25 °C. CONCLUSIONS: Our protocol significantly improves the efficiency of generative propagation of vanilla and allows for significantly higher germination percentages than previously described. The strongly positive effect of hydrochloric acid may be related to the adaptation of seeds to endozoochorous dispersal.

High-throughput Method for Novel Medium Development for Culture of Anaerobic Gut Bacteria.

Gut microbiota play important roles in the health of their host and detailed investigation of these organisms requires in vitro culture. Culturing strictly anaerobic bacteria can be a challenge as the gut environment they inhabit is nutritionally complex. Use of complex media containing nutritionally rich but undefined gut fluid reduces the accuracy of physiological and metabolomic studies. Here we present a high-throughput protocol for comparing growth rates of fastidiously anaerobic bacteria on different media. These protocols can be used to develop a solid medium made up of commercially sourced ingredients, providing replicable growth conditions for previously uncultured anaerobic bacteria. As many fastidious bacteria grow poorly in a liquid broth, these protocols measure bacterial growth rate on solid media. These protocols speed up and simplify the growth rate measurement process by using a multiwell format and equations in place of physical McFarland standards to calculate approximate cell density. Bacterial strains belonging to the families Erysipelotrichaceae and Lachnospiraceae (phylum Firmicutes) isolated from the hindgut of Kyphosus sydneyanus were used to demonstrate the efficacy of these protocols. Bacterial growth rates were compared between a nutritionally rich medium with gut fluid versus a novel replicable medium with mannitol. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of solid YCFA growth medium Basic Protocol 2: Collection of fish gut samples and plating to single isolates Basic Protocol 3: Genetic identification of single isolates with colony PCR and 16S rRNA gene sequencing Basic Protocol 4: Measurement of bacterial growth rates on solid media.

[Epigenetic changes in the promoter of the fragile histidine triad (FHIT) gene in human sebocytes under the influence of in vitro culture]. / Epigenetische Veränderungen am Promotor des fragilen Histidin-Trias(FHIT)-Gens in menschlichen Sebozyten unter dem Einfluss der In-vitro-Kultur.

BACKGROUND: Due to the lack of tumor suppressor function of the fragile histidine triad (FHIT) gene product, sebaceous gland carcinomas can develop. OBJECTIVE: The model of the sebocyte cell line SZ95 was used to identify methylated CpG islands at the 5'-end of the FHIT gene and the decrease of gene expression as well as the increase of double-stranded (ds) DNA breaks were examined. MATERIAL AND METHODS: Methylation, immunofluorescence analysis, promotor sequencing and treatment of SZ95 cells with 5­azacytidine/trichostatin A (TSA). RESULTS: The cultivation was accompanied by an increasing methylation of the CpG islands, a decrease of the FHIT gene expression and an accumulation of ds-DNA breaks. Treatment with 5­azacytidine/TSA showed a decrease in DNA methylation and a re-expression of FHIT transcripts. DISCUSSION: Epigenetic changes in the cellular genome are caused by in vitro cell culture. Consequently, a positive selection of sebocytes with an epigenetically inactivated FHIT locus occurs.

Biological Activity and NMR-Fingerprinting of Balkan Endemic Species Stachys thracica Davidov.

Stachys thracica Davidov is a Balkan endemic species distributed in Bulgaria, Greece, and Turkey. In Bulgaria, it is classified as "rare" and is under the protection of the Bulgarian biodiversity law. The aim of our study was to develop an efficient protocol for ex situ conservation of S. thracica and to perform comparative NMR-based metabolite profiling and bioactivity assays of extracts from in situ grown, in vitro cultivated, and ex vitro acclimated plants. Micropropagation of S. thracica was achieved by in vitro cultivation of mono-nodal segments on basal MS medium. Ex vitro adaptation was accomplished in the experimental field with 83% survival while conserved genetic identity between in vitro and ex vitro plants as shown by the overall sequence-related amplified polymorphism marker patterns was established. Verbascoside, chlorogenic acid, and trigonelline appeared the main secondary metabolites in in situ, in vitro cultivated, and ex vitro acclimated S. thracica. High total phenolic and flavonoid content as well as antioxidant and radical scavenging activity were observed in in situ and ex vitro plants. Further, the anti-inflammatory activity of S. thracica was tested by hemolytic assay and a high inhibition of the complement system was observed. Initiated in vitro and ex vitro cultures offer an effective tool for the management and better exploitation of the Stachys secondary metabolism and the selection of lines with high content of bioactive molecules and nutraceuticals.

Laboratory Selection of Trypanosomatid Pathogens for Drug Resistance.

The selection of parasites for drug resistance in the laboratory is an approach frequently used to investigate the mode of drug action, estimate the risk of emergence of drug resistance, or develop molecular markers for drug resistance. Here, we focused on the How rather than the Why of laboratory selection, discussing different experimental set-ups based on research examples with Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. The trypanosomatids are particularly well-suited to illustrate different strategies of selecting for drug resistance, since it was with African trypanosomes that Paul Ehrlich performed such an experiment for the first time, more than a century ago. While breakthroughs in reverse genetics and genome editing have greatly facilitated the identification and validation of candidate resistance mutations in the trypanosomatids, the forward selection of drug-resistant mutants still relies on standard in vivo models and in vitro culture systems. Critical questions are: is selection for drug resistance performed in vivo or in vitro? With the mammalian or with the insect stages of the parasites? Under steady pressure or by sudden shock? Is a mutagen used? While there is no bona fide best approach, we think that a methodical consideration of these questions provides a helpful framework for selection of parasites for drug resistance in the laboratory.

In Vitro Multiplication and NMR Fingerprinting of Rare Veronica caucasica M. Bieb.

Micropropagation of rare Veronica caucasica M. Bieb. was achieved by successful in vitro cultivation of mono-nodal segments on MS medium supplemented with 1.0 mg L-1 6-benzylaminopurine (BA) and then transferring the regenerated plants on hormone free basal MS medium for root development. In vitro multiplicated plants were successively acclimated in a growth chamber and a greenhouse with 92% survival. The number of plastid pigments and the total phenolics content in in vitro cultivated and ex vitro adapted plants were unchanged, and no accumulation of reactive oxygen species (ROS) was detected by staining with 3-3'-diaminobenzidine (DAB) and 2',7'-dichlorofluorescein diacetate (DCF-DA). Nuclear Magnetic Resonance (NMR) fingerprinting allowed for the identification of the major alterations in metabolome of V. caucasica plants during the process of ex situ conservation. Iridoid glucosides such as verproside, aucubin and catalpol were characteristic for in vitro cultivated plants, while in ex vitro acclimated plants phenolic acid-protocatechuic acid and caffeic acid appeared dominant. The successful initiation of in vitro and ex vitro cultures is an alternative biotechnological approach for the preservation of V. caucasica and would allow for further studies of the biosynthetic potential of the species and the selection of lines with a high content of pharmaceutically valuable molecules and nutraceuticals.

In vitro cultivation of Trypanosoma congolense bloodstream forms: State of the art and advances.

Animal African Trypanosomosis (AAT or Nagana) is a severe vector-borne disease caused by protozoan parasites belonging to the Trypanosomatidae family and is usually cyclically transmitted by blood-sucking tsetse flies. AAT remains a major problem in sub-Saharan Africa. Among the main AAT causative agents, Trypanosoma congolense (T. congolense or Tc) is one of the most important trypanosome species, in terms of economic and animal health impacts, infecting cattle and a wide range of animal hosts as well. To advance in AAT prevention and control, it is essential to better understand trypanosome biology and pathogenesis using bloodstream form (BSF) in vitro culture. The in vitro cultivation of T. congolense IL3000 BSF strain is already well established and widely used in research studies and drug activity assays. However, it may probably no longer truly reflect the reality of field trypanosome strains, due to decades of use and subsequent modifications. Here, we propose a novel culture protocol that supports the long-term in vitro growth of the animal-infective BSFs of three Savannah and Forest types of T. congolense strains, including T. congolense clone IL1180, which is not only a field strain but also a commonly-used reference strain in experimental animal assays. We established a homemade culture medium which made it possible to sustain T. congolense IL1180 growth from infected mouse blood for 18 days in axenic conditions. Moreover, we developed an efficient freezing/thawing system that allowed, for the first time, T. congolense IL1180 BSF growth within 30 days after thawing. Our results on T. congolense adaptation to in vitro culture are encouraging for future gene studies using new molecular tools or for new therapeutic drug assays.

The Expression Pattern of Surface Markers in Canine Adipose-Derived Mesenchymal Stem Cells.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1-P3) to late (P4-P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73's one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.