Oscar mejlar över en helt ny studie (31/5) med toppaktuella ämnet Regenerativ medicin.
First published: 31 May 2022
Abstract
Living interfaces are established as a novel class of active materials that aim to provide an alternative to traditional static cell culture methods by enabling users to accurately control cell behaviour in a precise, dynamic, and reliable system-internal manner. To this day, the only reported biointerface has been a coculture between a biofilm of nonpathogenic genetically engineered bacteria and mammalian cells, where the recombinant proteins produced by the bacteria directly influence cell behaviour. In this work, a biointerface is presented between Lactococcus lactis (L. lactis) and human mesenchymal stem cells (hMSCs). L. lactis have been engineered to produce human C-X-C motif chemokine ligand 12, thrombopoietin, vascular cell adhesion protein 1, and the 7th–10th type III domains of human fibronectin, with the aim of recreating the native bone marrow conditions ex vivo. This active microenvironment has been shown to maintain key hMSC stemness markers, preventing their osteogenic and adipogenic differentiation, and maintaining high stem cell viability and physiological cell-to-substrate adhesion dynamics. This work presents proof of concept data that hMSC stemness can be regulated by living materials, using a system based on the symbiotic interaction between different engineered bacteria and mammalian cells.
1 Introduction
The growing demand for novel and efficient therapeutic methods to treat complex clinical conditions has created the need for new approaches in regenerative medicine. Tissue engineering is a field of growing scientific interest due to its interdisciplinary approach of combining the physical and biological characteristics of human tissues with the principles of engineering and materials science.
The possibility of exploiting the material properties of biocompatible polymers and scaffolds to create and mimic a microenvironment for natural tissue growth could be used in a variety of clinical settings such as transplant development or organ regeneration.
Human mesenchymal stem cells (hMSCs), also known as multipotent mesenchymal stromal cells are a key player in tissue engineering because of their remarkable potential to differentiate into a variety of cell types when stimulated with the appropriate biochemical, physical and mechanical cues. This has placed MSCs in the spotlight of regenerative medicine research and has created the need to develop strategies in order to control the cells’ fate in a precise and reproducible manner.Despite the wide variety of literature reporting hMSC differentiation studies, efforts to precisely control the differentiation of these cells have been impaired due to the complexity of the proposed systems.
5. Experimental Section ( HoloMonitorverifiering)
Assessment of Recombinant Protein Bioactivity
The evaluation of CXCL12 bioactivity was conducted by studying the motility dynamics of human MSCs. The stem cells were cultured overnight in sterile 24-well tissue culture plates. The following day, a 4 h culture of CXCL12-expressing L. lactis was conducted in DMEM, to pre-condition the media for the motility study. Following the culture, the hMSCs were incubated in DMEM conditioned with L. lactis-secreted or commercial CXCL12 (PeproTech), both at a concentration of 200 ng mL−1, while DMEM without any cytokine was used as a control. Stem cell mobility was assessed by live phase holographic imaging, using a HoloMonitor M4 microscope (PHI). The cells were monitored for 24 h using label-free imaging and their motility was quantified by measuring the nondirectional movement and speed of the cells in our different samples. The average cell speed was automatically determined by applying the HoloMonitor Cell Motility Assay on the recorded time-lapse sequence.
hMSC Tracking
Bone marrow derived hMSCs were cultured overnight on prepared L. lactis biofilms. Depending on the needs of each experimental setup, the hMSCs were tracked continuously for 1 or 24 h using time-lapse microscopy. During the measurements, the cells were maintained in hMSC maintenance media, at a humidified incubator at 37 °C, 5% CO2.
Figure S6. Characterisation of the biological activity of the CXCL12 and TPO expressed by L. lactis. In 6A and 6B, human bone marrow-derived mesenchymal stem cells were cultured for 24h in DMEM supplemented with either commercial human recombinant CXCL12 (Peprotech) at 200 ng mL-1 , preconditioned with L. lactis expressing CXCL12 or L. lactis EMPTY, that is, bacteria that does not express any heterologous protein. Negative control refers to DMEM medium alone without any supplements. Initially, the cells were cultured overnight until they adhered to the glass substrate. After 16h, the media was substituted with either the media preconditioned with the L. lactis culture, supplemented with commercial recombinant CXCL12, or bacterial and commercial media negative controls. Cells were imaged every 10 minutes for a total of 24 h in a HoloMonitor M4 digital time-lapse cytometer located inside a cell culture incubator at 37°C, 5% CO2 humidified atmosphere and analysed afterwards in the App Suite cell imaging software to determine the instantaneous and average migration speed over 24h. 6A shows the average migration speed in µm/h for the four samples for the 24 h time period. The graph shows a LOWESS 20-point averaged spline for clarity. 6B shows the average speed over the 24 h accumulated time period. Data shows a statistically significant difference between the hMSC cultured on the L. lactis-CXCL12 preconditioned medium, the medium supplemented with commercial CXCL12 and the negative controls. It is interesting to note that there is no statistically significant difference between the commercial and the L. lactis-expressed CXCL12, suggesting that the bacterially produced CXCL12 is biologically active. Data was analysed using a one-way parametric ANOVA with α=0.05 and Tukey’s post-hoc test. ns = non-significant (p = 0.0756), * p < 0.05, **** p < 0.0001.
4 Conclusion
Here, we demonstrate the potential of genetically engineered living biointerfaces to actively control stem cell behavior. Despite the number of living materials already developed, our work is based on a radical, multifaceted approach in combining the active, self-regulatory element of the biointerface, a variety of recombinant cytokines and adhesion factors produced by the bacteria and 2D and 3D culture conditions to produce novel living materials. Our approach benefits from the nonpathogenic nature of L. lactis, providing our system with a significant advantage over current approaches on living materials based in E. coli, and providing a promising potential for this work to be expanded and used in future clinical applications. We have demonstrated hMSCs can be successfully cultured on L. lactis biofilms, retaining high viability and displaying adhesion dynamics and cell movement comparable to traditional culture methods. Our work also provides proof that L. lactis can be used as a substrate to actively control hMSC behavior, retaining the cells in a naïve state in long term cultures, without affecting their differentiation potential. This new living material can be easily tuned and adjusted to a variety of applications, by instructing different recombinant protein expression or cocultures with different cell types. Our vision is that our platform will be used in further studying different aspects of stem cell fate decisions for medical applications and even in adapted versions as a clinical tool for tissue engineering applications.
Ur PHI`s synvinkel är detta en bonusinfo. Det för att HoloMonitor visar sin användbarhet innan själva processen att bygga vävnad tar vid. Det är just i fasen vävnadsbyggnad HoloMonitor har sin styrka,att vara kontrollorgan under hela processen och slutligen som en godkännandefaktor att visa upp för en myndighet ett RM-läkemedelstillverkande företag behöver ha tillstånd från.
Men nu visar forskarna hur oundgänglig tekniken är redan i grundfasen,RM-forskningen.Ett mkt bra signalvärde för andra labb som sysslar med RM-grundforskning.
Bloggen har skrivit att utvecklingen går fort inom den Regenerativa medicinen. Denna studie visar på det och driver även utvecklingen än snabbare framåt.
Ps. Sprid gärna denna info vidare. Den är förbaskat bra. Ds
Mvh the99
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