Reversible Secretion of Glycosaminoglycans and Proteoglycans by Cyclically Stretched Valvular Cells in 3D Culture

Sarcomas are tumours of mesenchymal origin, which can arise in bone or soft tissues. They are rare but frequently quite aggressive and with a poor outcome. New approaches are needed to characterise these tumours and their resistance mechanisms to current therapies, responsible for tumour recurrence and treatment failure. This review is focused on the potential of three-dimensional (3D) in vitro models, including multicellular tumour spheroids (MCTS) and organoids, and the latest data about their utility for the study on important properties for tumour development. The use of spheroids as a particularly valuable alternative for compound high throughput screening (HTS) in different areas of cancer biology is also discussed, which enables the identification of new therapeutic opportunities in commonly resistant tumours.

Download Full-text

Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0214 ◽

2017 ◽

Vol 28 (8) ◽

pp. 1088-1100 ◽

Cited By ~ 2

Author(s):

Lynne A. Lapierre ◽

Elizabeth H. Manning ◽

Kenya M. Mitchell ◽

Cathy M. Caldwell ◽

James R. Goldenring

Keyword(s):

Mdck Cells ◽

Three Dimensional ◽

3D Culture ◽

Cyst Formation ◽

Epithelial Polarity ◽

Lateral Membrane ◽

Low Calcium ◽

Single Lumen ◽

Apical Junctions ◽

Temporal Localization

MARK2 regulates the establishment of polarity in Madin–Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)–expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.

Download Full-text

Comparative evaluation of osteogenic differentiation potential of stem cells derived from dental pulp and exfoliated deciduous teeth cultured over granular hydroxyapatite based scaffold

BMC Oral Health ◽

10.1186/s12903-021-01621-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Manal Nabil Hagar ◽

Farinawati Yazid ◽

Nur Atmaliya Luchman ◽

Shahrul Hisham Zainal Ariffin ◽

Rohaya Megat Abdul Wahab

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

3D Culture ◽

Permanent Teeth ◽

Deciduous Teeth ◽

Osteogenic Potential ◽

Control Group ◽

Rt Pcr ◽

Differentiation Potential

Abstract Background Mesenchymal stem cells isolated from the dental pulp of primary and permanent teeth can be differentiated into different cell types including osteoblasts. This study was conducted to compare the morphology and osteogenic potential of stem cells from exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC) in granular hydroxyapatite scaffold (gHA). Preosteoblast cells (MC3T3-E1) were used as a control group. Methodology The expression of stemness markers for DPSC and SHED was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). Alkaline phosphatase assay was used to compare the osteoblastic differentiation of these cells (2D culture). Then, cells were seeded on the scaffold and incubated for 21 days. Morphology assessment using field emission scanning electron microscopy (FESEM) was done while osteogenic differentiation was detected using ALP assay (3D culture). Results The morphology of cells was mononucleated, fibroblast-like shaped cells with extended cytoplasmic projection. In RT-PCR study, DPSC and SHED expressed GAPDH, CD73, CD105, and CD146 while negatively expressed CD11b, CD34 and CD45. FESEM results showed that by day 21, dental stem cells have a round like morphology which is the morphology of osteoblast as compared to day 7. The osteogenic potential using ALP assay was significantly increased (p < 0.01) in SHED as compared to DPSC and MC3T3-E1 in 2D and 3D cultures. Conclusion gHA scaffold is an optimal scaffold as it induced osteogenesis in vitro. Besides, SHED had the highest osteogenic potential making them a preferred candidate for tissue engineering in comparison with DPSC.

Download Full-text

Type II Collagen-Conjugated Mesenchymal Stem Cells Micromass for Articular Tissue Targeting

Biomedicines ◽

10.3390/biomedicines9080880 ◽

2021 ◽

Vol 9 (8) ◽

pp. 880

Author(s):

Shamsul Bin Sulaiman ◽

Shiplu Roy Chowdhury ◽

Mohd Fauzi Bin Mh Busra ◽

Rizal Bin Abdul Rani ◽

Nor Hamdan Bin Mohamad Yahaya ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Targeted Delivery ◽

Expression Profiles ◽

3D Culture ◽

Type Ii Collagen ◽

Cartilage Defects ◽

Target Tissue ◽

Type Ii ◽

Antibody Conjugation

The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis.

Download Full-text

Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

International Journal of Molecular Sciences ◽

10.3390/ijms22052491 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2491

Author(s):

Yujin Park ◽

Kang Moo Huh ◽

Sun-Woong Kang

Keyword(s):

Cell Culture ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Accurate Method ◽

New Drugs ◽

Toxicity Screening ◽

Cellular Functions ◽

Toxicity Of Drugs ◽

External Stimuli

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.

Download Full-text

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury

The International Journal of Artificial Organs ◽

10.1177/0391398820986809 ◽

2021 ◽

pp. 039139882098680

Author(s):

Xuefeng Zhang ◽

Nan Wang ◽

Yuhua Huang ◽

Yan Li ◽

Gang Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Extracellular Vesicles ◽

Therapeutic Potential ◽

Expression Profiles ◽

Ischemia Reperfusion ◽

Three Dimensional ◽

3D Culture ◽

Placental Mesenchymal Stem Cells ◽

2D And 3D

Background: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). Methods: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. Results: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. Conclusion: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

Download Full-text

Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22063042 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3042

Author(s):

Eun Ju Lee ◽

Khurshid Ahmad ◽

Shiva Pathak ◽

SunJu Lee ◽

Mohammad Hassan Baig ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Cell Adhesion ◽

3D Culture ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Primary Cells ◽

Cell Culture Techniques ◽

Culture Techniques ◽

Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Download Full-text

3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential

Journal of Personalized Medicine ◽

10.3390/jpm11060528 ◽

2021 ◽

Vol 11 (6) ◽

pp. 528

Author(s):

Spoorthi Ravi Banavar ◽

Swati Yeshwant Rawal ◽

Shaju Jacob Pulikkotil ◽

Umer Daood ◽

Ian C. Paterson ◽

...

Keyword(s):

Stem Cells ◽

Periodontal Ligament ◽

3D Culture ◽

Osteogenic Potential ◽

Culture Methods ◽

Periodontal Ligament Stem Cells ◽

Raman Analysis ◽

Atp Production ◽

Tnf Α ◽

Inflammatory Milieu

Background: The effects of lipopolysaccharide (LPS) on cell proliferation and osteogenic potential (OP) of MSCs have been frequently studied. Objective: to compare the effects of LPS on periodontal-ligament-derived mesenchymal stem cells (PDLSCs) in monolayer and 3D culture. Methods: The PDLSCs were colorimetrically assessed for proliferation and osteogenic potential (OP) after LPS treatment. The 3D cells were manually prepared by scratching and allowing them to clump up. The clumps (C-MSCs) were treated with LPS and assessed for Adenosine triphosphate (ATP) and OP. Raman spectroscopy was used to analyze calcium salts, DNA, and proline/hydroxyproline. Multiplexed ELISA was performed to assess LPS induced local inflammation. Results: The proliferation of PDLSCs decreased with LPS. On Day 28, LPS-treated cells showed a reduction in their OP. C-MSCs with LPS did not show a decrease in ATP production. Principal bands identified in Raman analysis were the P–O bond at 960 cm−1 of the mineral component, 785 cm−1, and 855 cm−1 showing qualitative changes in OP, proliferation, and proline/hydroxyproline content, respectively. ELISA confirmed increased levels of IL-6 and IL-8 but with the absence of TNF-α and IL-1β secretion. Conclusions: These observations demonstrate that C-MSCs are more resistant to the effects of LPS than cells in monolayer cell culture. Though LPS stimulation of C-MSCs creates an early pro-inflammatory milieu by secreting IL-6 and IL-8, PDLSCs possess inactivated TNF promoter and an ineffective caspase-1 activating process.

Download Full-text