seeding density
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2022 ◽  
Vol 198 ◽  
pp. 104701
Author(s):  
Christopher J. Lortie ◽  
Nargol Ghazian ◽  
Mario Zuliani

2022 ◽  
Vol 5 (1) ◽  
pp. 8
Author(s):  
Giorgia Borciani ◽  
Giorgia Montalbano ◽  
Nicola Baldini ◽  
Chiara Vitale-Brovarone ◽  
Gabriela Ciapetti

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.


2021 ◽  
Vol 130 (22) ◽  
pp. 225302
Author(s):  
Anupam K. C. ◽  
Rony Saha ◽  
Jonathan Anderson ◽  
Anival Ayala ◽  
Christopher Engdahl ◽  
...  

2021 ◽  
Vol 8 (12) ◽  
pp. 209
Author(s):  
John P. Soleas ◽  
Linwen Huang ◽  
Elisa D’Arcangelo ◽  
Maria Cristina Nostro ◽  
Thomas K. Waddell ◽  
...  

Chemically directed differentiation of pluripotent stem cells (PSCs) into defined cell types is a potent strategy for creating regenerative tissue models and cell therapies. In vitro observations suggest that physical cues can augment directed differentiation. We recently demonstrated that confining human PSC-derived lung progenitor cells in a tube with a diameter that mimics those observed during lung development results in the alteration of cell differentiation towards SOX2−SOX9+ lung cells. Here we set out to assess the robustness of this geometric confinement effect with respect to different culture parameters in order to explore the corresponding changes in cell morphometry and determine the feasibility of using such an approach to enhance directed differentiation protocols. Culture of progenitor cells in polydimethylsiloxane (PDMS) tubes reliably induced self-organization into tube structures and was insensitive to a variety of extracellular matrix coatings. Cellular morphology and differentiation status were found to be sensitive to the diameter of tube cells that were cultured within but not to seeding density. These data suggest that geometric cues impose constraints on cells, homogenize cellular morphology, and influence fate status.


2021 ◽  
Vol 58 (2) ◽  
pp. 209-219
Author(s):  
Piotr Szulc ◽  
Katarzyna Ambroży-Deręgowska

Summary In this paper, we investigate the effect of seeding density on several morphological features such as plant height, height of the production ears, ear length, ear diameter, leaf area, and LAI (leaf area index). Inference is based on a series of three-year two-factor experiments with two hybrid maize varieties – SY Cooky and Drim “stay-green” type – and 5 sowing densities: 6, 7, 8, 9 and 10 plants per m2. The “stay-green” maize variety had production cobs significantly higher on the plant, and had a thicker cob and a larger leaf assimilation area than the conventional variety. Increasing maize sowing density from 6 to 10 plants m−2 resulted in a linear decrease in cob length and diameter, while it increased the LAI. Significantly higher chlorophyll content, expressed in SPAD units, was found in the “stay-green” hybrid at the BBCH 67 stage in a wet (2012) and drier year (2014), compared with the traditional variety. This may indicate that such a variety is more tolerant to stress conditions.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander P Browning ◽  
Jesse A Sharp ◽  
Ryan J Murphy ◽  
Gency Gunasingh ◽  
Brodie Lawson ◽  
...  

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.


Author(s):  
Daniel Wohlfarth ◽  
Veronika Frehtman ◽  
Marcus Müller ◽  
Martin Vogel ◽  
Linh Minh Phuc Phan ◽  
...  

Abstract The oncolytic virus H-1PV is a promising candidate for various cancer treatments. Therefore, production process needs to be optimized and scaled up for future market release. Currently, the virus is produced with minimum essential medium in 10-layer CellSTACK® chambers with limited scalability, requiring a minimum seeding density of 7.9E3 cells/cm2. Production also requires a 5% fetal bovine serum (FBS) supplementation and has a virus yield up to 3.1E7 plaque-forming units (PFU)/cm2. Using the animal-free cell culture medium VP-SFM™ and a new feeding strategy, we demonstrate a yield boost by a mean of 0.3 log while reducing seeding density to 5.0E3 cells/cm2 and cutting FBS supplementation by up to 40% during the production process. Additionally, FBS is completely removed at the time of harvest. Eleven commercial micro- and macrocarriers were screened regarding cell growth, bead-to-bead transfer capability, and virus yield. We present a proof-of-concept study for producing H-1PV on a large scale with the microcarrier Cytodex® 1 in suspension and a macrocarrier for a fixed-bed iCELLis® bioreactor. A carrier-based H-1PV production process combined with an optimized cell culture medium and feeding strategy can facilitate future upscaling to industrial-scale production. Key points • Virus yield increase and FBS-free harvest after switching to cell culture medium VP-SFM™. • We screened carriers for cell growth, bead-to-bead transfer capability, and H-1PV yield. • High virus yield is achieved with Cytodex® 1 and macrocarrier for iCellis® in Erlenmeyer flasks.


Land ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1186
Author(s):  
Hong Li ◽  
Min Zhao ◽  
Changhui Peng ◽  
Haiqiang Guo ◽  
Qing Wang ◽  
...  

Although rice paddy fields are one of the world’s largest anthropogenic sources of methane CH4, the budget of ecosystem CH4 and its’ controls in rice paddies remain unclear. Here, we analyze seasonal dynamics of direct ecosystem-scale measurements of CH4 flux in a rice-wheat rotation agroecosystem over 3 consecutive years. Results showed that the averaged CO2 uptakes and CH4 emissions in rice seasons were 2.2 and 20.9 folds of the wheat seasons, respectively. In sum, the wheat-rice rotation agroecosystem acted as a large net C sink (averaged 460.79 g C m−2) and a GHG (averaged 174.38 g CO2eq m−2) source except for a GHG sink in one year (2016) with a very high rice seeding density. While the linear correlation between daily CH4 fluxes and gross ecosystem productivity (GEP) was not significant for the whole rice season, daily CH4 fluxes were significantly correlated to daily GEP both before (R2: 0.52–0.83) and after the mid-season drainage (R2: 0.71–0.79). Furthermore, the F partial test showed that GEP was much greater than that of any other variable including soil temperature for the rice season in each year. Meanwhile, the parameters of the best-fit functions between daily CH4 fluxes and GEP shifted between rice growth stages. This study highlights that GEP is a good predictor of daily CH4 fluxes in rice paddies.


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