Lab-on-Chip Culturing System for Fungi—Towards Nanosatellite Missions

Round 1

Reviewer 1 Report

see attached file.

Comments for author File: Comments.pdf

Author Response

Responses to Reviewers’ comments:

1. It is recommended to mention about the applications of this study at the end of abstract: ''The findings of this study can help for better understanding of …''. Meanwhile, it is suggested to discuss more about the findings of this study in the abstract.

Ad.1. We thank the Reviewer for that comment. The abstract section was completed with the description of our findings utility:

“The findings of this study can provide substantial new knowledge on microscopic fungi cultivation in lab-on-chip devices, other soil organisms, as well as a potential behavior of these species in microgravity conditions. Culturing system shown in this work can help mycologists to provide better understanding of microscopic fungi nature and their development mechanisms at a single spore level. This opens the way towards regular usage of microfluidic tools in agriculture and horticulture fields and more importantly, in future research on microscopic fungi in space, e.g. as a part of nanosatellite missions.”

2. In the introduction, I would suggest to clearly motivate the different shapes of the LOC. What are the shapes that have been chosen relevant? Where can they be found, and what are applications? As much work has been done already in this research field, it is very important to emphasize the novelty and potential relevance of this study in the manuscript.

Ad. 2. Cell culturing LOCs are typically equipped with microchamber(s) – area for cells growth and microchannel(s), passing through the microchamber(s) and delivering the culturing media for cells feeding. Depending on the technology employed for the lab-on-chip fabrication, some limitations in LOC construction may appear, which can simply modify the ultimate microchamber and microchannel geometry. Other issue is the fact that every microscopic object, e.g. fungi species Fusarium culmorum, has its own characteristics, that may simply determine the most appropriate chip design. The literature on the subject is rich in examples of cell culturing LOCs fabricated out of polymers (e.g. PDMS, COC, PMMA), glass (borosilicate, FOTURAN), as well as utilizing 3D printing method; nevertheless, the papers only to a small extent mention about the LOCs dedicated to fungi cultivation (please see the detailed description in the Introduction section of this paper). Moreover, these LOCs are made of PDMS solely, which based on recent literature reports and investigation provided by the authors, may face some problems with biocompatibility [1-3]. For instance, leaching of uncured oligomers from the polymer network to the culture medium may badly affect the cells, making cell culture unreliable [1]. For this reason, in this paper the authors decided to employ all-glass technology for the chip fabrication. Apart from the substantial experience of the group in this regard, covering development of many structures dedicated to culturing of radically different bio-objects (e.g. freshwater microorganisms, animal oocytes, human cancer and normal cells) [4-6], the preliminary research on LOC design for microscopic fungi was recently done and presented in the paper [6]. However, the aforementioned research did not include microflow culture – only stationary culture conditions were applied. On that basis, other research had to be undertaken to propose to best LOC design for F. culmorum fungi species, assuming powdered fungi form obtainment (necessary to ensure delayed experimentation on fungi, e.g. due to long waiting for the rocket launch), and autonomous media delivery system. The idea to propose the structure including separately gas and media channel connected by the matrix of connecting channels was created based on the microscopic observations, showing that in our preliminary design (similar as shown in the paper [6]), fungi spores do not grow directly in water environment, but rather group in dryer microchannel parts, being a side effects of glass etching process. On that basis, the structure containing independent – dry microchannel, but connected with medium microchannel and thus, maintaining appropriate humidity environment, was proposed, modelled, tested and validated. As the translation of macroscale methodologies towards microscale lab-on-chip approach is always not trivial, the matter of dry, “non-immersed” fungi spores is a significant novelty, which may open the way towards other applications, suggesting indirect creation of chemical gradients within the culturing habitat. Moreover, use of all-glass technology is still unpopular with scientists dealing with microfluidics, thus we hope that this work is a good example of its application, which may shortly change the attitude to glass microengineering, by and large. However, as mentioned earlier in this point, every bio-object and experiment type, requires special scientific and technological insight, thus the structure successfully implemented herein for Fusarium culmorum culture, will not necessarily meet the demands for culturing of other microscopic fungi species.

This description was added to the section “2.3. LOC – construction and technology”.

References:

[1] K.J. Regehr, et al., Biological implications of polydimethylsiloxane-based microfluidic cell culture, Lab Chip, vol. 9, pp. 2132–2139, 2009

[2] S. Halldorsson, E. Lucumi, R. Gómez-Sjöberg, R.M. Fleming, Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices, Biosens. Bioelectron, vol. 63, pp. 218–231, 2015

[3] R. Mukhopadhyay, When PDMS isn’t the best, Anal. Chem., vol. 79, pp. 3249–3253, 2007

[4] 54. A. Podwin, W. Kubicki, J. Dziuban, Study of the behavior of Euglena viridis, Euglena gracilis and Lepadella patella cultured in all-glass microaquarium, Biomedical Microdevices, vol. 19, pp. 1-10, 2017

[5] D. Przystupski, A. Górska, O Michel, A. Podwin, P. Sniadek, R. Łapczynski, J. Saczko, J. Kulbacka, Testing Lab-on-a-Chip Technology for Culturing Human Melanoma Cells under Simulated Microgravity, Cancers, vol. 13, 402, 2021

[6] 55. A. Podwin, T. Janisz, K. Patejuk, P. Szyszka, R. Walczak, J. Dziuban, Towards microfluidics for mycology – material and technological studies on LOCs as new tools ensuring investigation of microscopic fungi and soil organisms, Bulletin of the Polish Academy of Sciences. Technical Sciences, pp. 1-9, 2021

3. The literature survey is not complete. A look at the list of references reveals that practically all of them cover the period 2005-2017 except one reference that dates back from 2020. Regarding the history of the topic covered by the authors, this list of references cannot be adequate and a number of important references is necessarily missing. For example, references related to work by Ashrafizadeh et al. on interfacial hydrodynamic behavior of LOC under electrokinetic conditions is missing. Also, key aspects on the functioning of electrified lab on disc systems are not mentioned, see e.g. Biosensors and Bioelectronics (2022): 114381; Electrochimica Acta (2022): 141175; Langmuir 38.33 (2022): 10313-10330.; Physical Chemistry Chemical Physics 24.34 (2022): 20303-20317.

Ad. 3. We thank the Reviewer for that comment. Certainly, the literature survey was lacking the latest articles in the field. Positions provided by the Reviewer, as well as additional papers (Y.-S. Lee, Y.-T. Lu, C.-M. Chang, C.-H. Liu, Finger-powered cell-sorting microsystem chip for cancer-study applications, Sensors and Actuators B: Chemical, vol. 370, 132430, 2022 and J.J. Feng, S. Hedtrich, A similarity scaling approach for organ-on-chip devices, Lab Chip, vol. 22, pp. 3663-3667, 2022) were added to the Introduction and section entitled “2.3. LOC – construction and technology. “ 

4. The authors need to better explain the nature of the force driving the hydrodynamic flow and, more generally, the level of information on adopted boundary conditions is not sufficient in the text.

Ad. 4. The equations solved by this module are the Navier-Stokes equations for conservation of momentum and the continuity equation for conservation of mass. The definition of laminar flow based on the Reynolds number was also used. The following boundary conditions were adopted: at the inlet of the liquid channel (according to the Figure 1), the liquid flow velocity was 200 µL/min, which was set in accordance with the planned in the experiment, while an atmospheric pressure was established at the outlet of the liquid channel and at the inlet and outlet of the gas channel. Water was chosen as the liquid and air as the gas. Next, the project was meshed. A uniform mesh for the entire model was used (Fig. 2). As a result, it could be observed that the spread of the liquid in the medium channel was as expected (Fig. 3a). At the same time, the correct working of the channels connecting both chambers was confirmed, the task of which was to provide humidity in the gas chamber, but not to flood it with nutrient solution. The gas channel remained almost dry (Fig. 3b).

The description concerning the Comsol numerical simulations was improved and all the aforementioned data was attached to the manuscript file.

5. The authors must relate the complexity of the adopted channels to existing technology or at least explain whether or not the LOC they theoretically construct is realistic given the current practice in membrane synthesis and fabrication. Computing things is one thing, relate them to experimental reality is another. The paper in its current form critically misses any attempt to connect these two aspects. Efforts by the authors in their manuscript should be done to provide the reader elements that motivate acceptability of their theoretical computations as related to current practice.

Ad. 5. As mentioned in the Ad. 2, the idea to propose the structure including separately gas and media channel connected by the matrix of connecting channels was created based on the microscopic observations, showing that in our preliminary chip design (similar as shown in the paper [6]), fungi spores do not grow directly in water environment, but rather group in dryer microchannel parts, being a side effects of glass etching process. On that basis, the structure containing independent – dry microchannel, but connected with medium microchannel and thus, maintaining appropriate humidity environment, was proposed, modelled, tested and validated.

The Authors cannot agree that the LOC design proposed in this manuscript is complex. Microengineering technologies have been developed for years (from about 3 decades) to ensure fabrication of glass structures of different geometries, characterized by shallow, even 1 µm-deep (or less) microchannels. As shown in the Fig. 5 and Fig. 9, real view of the all-glass LOC structure corresponds to our design entirely, which is related to possibility of precise forming of spatial structures based on wet chemical etching in HF acids. The matter of creation of different chemical gradients within LOC structures is also a common strategy, described in the recent literature widely (please see the references provided in the manuscript [58-60]), thus, opportunity to influence one of the microchannel and its environment utilizing other microchannel and shallow capillaries can be provided based on microfluidic techniques. The mechanisms of laminar flow which is faced within the microscale structures, as well as diffusion-based liquid mixing, can be treated as both advantageous and disadvantageous. In the case of micromixers –diffusion-based microfluidic mixing is highly insufficient, for cell culturing field in turn, these phenomena are rather beneficial, since they provide indirect, time-dependent microhabitat stimulation.

The literature cited in the article presents the construction of lab-chips using PDMS membranes ([59-60] in the manuscript). However, the experience of the authors of this article shows that this material is not fully biocompatible, especially if a long cultivation time is needed. Another aspect is choosing a material that could be used in outer space. The above necessities determined that the best option was to choose a glass material. The glass is not permeable to gases, thus it was necessary to propose a new geometry that would simultaneously provide the nutrient solution and, on the other hand, provide an appropriate gas atmosphere. Due to the above necessities, it is not possible to directly compare the literature data based on semi-permeable membranes with PDMS with the proposed own construction.

The article presents a comprehensive approach to the implementation of microfluidic lab-chips. The first stage was the design process. In the second stage, the structure was modeled in order to verify its correct operation. The last, third stage, presents the technological processes performed and the study of the work of the lab-chip in real conditions. Thus, the presented process of creating a lab-chip includes both computational and experimental aspects.

6. Critical commentary is needed from the author who eventually recommends directions for further research.

Ad. 6. We thank the Reviewer for that comment. The conclusion section was revised and potential future directions were outlined. Moreover, the significance of the numerical analysis was emphasized. 

“With a view to the aforementioned results, it can be assumed that our lab-on-chip platform can be used by mycologists in other laboratory studies (e.g. concerning environmental approaches and coculture investigation with plants or soil organisms), but also as a tool for astrobiological research, e.g. conducted in a CubeSat in LEO. This undoubtedly opens new horizons for LOCs utility in agriculture and horticulture fields, making the creation of controlled environments on-chip vital and of indispensable need, especially in the context of single spore insight. Microfluidic approach towards precise investigation of microscopic fungi and other soil dwellings can provide new knowledge on interactions of these species and their mutual interplay (symbiotic/parasitic relations). Mycorrhiza and its mechanisms play an important role in plant cultivation, thus further research in this field can bring new advancement, especially needed with a view to our potential colonization aspirations. Use of lab-on-chip techniques fits the demands of the aforementioned subjects. In addition, numerical modelling of the LOC structures with microflow management is a good practice that performed prior to experimentation can answer most of the scientific questions and describe microfluidic performance thoroughly. Computational work, as shown in this paper, ensures to evaluate the microflow consistency, remaining the LOC functionalities as assumed.” 

7. What are the advantages and disadvantages of this study? What are the limitations of this study? I recommend the authors to highlight these topics.

Ad. 7. The thank the Reviewer for that comment. Generally, this work is rather advantageous  than disadvantageous to the both engineering and biomedical fields. Moreover, it can be also of great interest for space biology research. Please, find the advantages and disadvantages of the work pointed below:

The main advantages of this work are:

- detailed literature analysis in the context of lab-on-chip technology, mycology investigation, space biology directions, as well as numerical modelling applications (thanks to the Reviewer recommendations),

- mentioning about the problems that can be faced with a view to preparation of the biological samples for astrobiological mission,

- methodology developed for achievement of the powdered fungi form directly on-chip,

- successful fabrication of the unique construction of the all-glass lab-on-chip, fulfilling the demands for culturing of sensitive biological objects (microscopic fungi), which performance was at first verified based on numerical modelling,

- description of the culturing system items and parameters of the microflow that were applied to assure long term culturing of fungi,

- successful culturing of fungi on-chip (visible mycelium growth) and interestingly, notable differences observed between the standard cultures and cultures performed in simulated microgravity,

- based on the works presented in this paper, the first European biological nanosatellite mission was undertaken and launched in January, this year.

The main disadvantages of this work are:

- technology of glass and related microengineering techniques are relatively complex, requiring multistep procedure, thus experienced scientist is needed to fabricate the microfluidic structures,

- RWV (Rotary Wall Vessel) is a single axis microgravity simulator, thus, the further research with the use of RPM (Random Position Machine) would be beneficial to imitate the microgravity conditions more reliably in the context of fungi growth,

- lack of quantitative analysis of fungi growth, but this information was provided in the revised manuscript file, based on suggestion of other Reviewer.

8. It is recommended to include a paragraph at the end of introduction to present the steps of the work like: ‘’First, the problem is formulated. Then, ... ‘’

Ad. 8. In reference to your recommendation, such a paragraph was included at the end of the Introduction section:

“The paper covers multiple steps that had to be undertaken to fulfil the assumptions of the work. At first, the selection of the biological object was done, with a focus put on its features and morphology. Next, the lab-on-chip structure, equipped with specific microchannels arrangement, was proposed, designed and numerically modelled to evaluate the LOC performance. Afterwards, the technological works were employed to obtain the lab-on-chip structure and then, the culturing system was assembled. The article is finished with cell culturing experiments performed in standard laboratory conditions and in simulated microgravity, imitated utilizing Rotary Wall Vessel.”

9. In the modeling portion of the paper, governing equations and boundary conditions should be included, and also, please provide a schematic of the boundary conditions.

Ad. 9. The thank for that comment. Please, see the Response to this question in the Ad. 4.

10. With respect to the numerical modeling, could you provide an example of the mesh independence test in the supplementary material?

Ad. 10. We thank the Reviewer for that comment – the tests on mesh independence were provided in the manuscript file (please see the Table 1 and additional description).

“We also carried out simulations with the use of three different number of mesh elements (coarse, normal, fine) at which was measured the point of flow in the liquid and gas channel (Table 1). It can be observed that the increasing number of mesh elements did not have a significant effect on the final result.”     

Table. 1: Comparison of flows in the channels depending on the mesh used

Number of mesh elements	Flow in liquid channel [m3/s]	Flow in gas channel [m3/s]
Coarse - 304 757	4,89441e-9	1,56306e-13
Normal - 626 127	4,85098e-9	1,55514e-13
Fine - 1 332 789	4,98822e-9	1,57639e-13

11. The conclusions section is badly missing perspectives. Conclusion section currently sticks to a summary of key findings of the study without any attempt to define relevant research directions motivated by the conclusions drawn by the authors from their computational work.

Ad. 11. That is right, the response to this comment is included in the Ad. 6.

12. There is insufficient inset in Figs. 2 and 3 and the data on the axis is not visible; please modify. Also it is recommended to add minor ticks (or intervals) on horizontal and vertical axis of data plots.

Ad. 12. Certainly, the inset of the mentioned figures was not clearly visible. Thus, the Fig. 2 and Fig. 3 was improved in the manuscript file. 

13. Please edit the language carefully, fix typos, and correct grammatical errors. Additionally, the layout of references should be thoroughly revised. Please see the newly published papers on “Applied Sciences”.

Ad. 13. We thank the Reviewer for that comment. The manuscript was carefully red and checked in the context of typos and other errors. Reference style was also revised.

 

Reviewer 2 Report

The authors have worked on fabricating a lab-on-chip device that can do fungal culture and be attached to a system to simulate microgravity. It is an exciting work with promising results. The paper can be accepted after including the following changes needed.

1. Figure 9 which shows fungal growth in the gas channel is not clear. Authors can provide an outline in the big image to make the inset more clear.

2. Comsol simulation shows no flow in the gas chamber where the fungi grow. So how are the fungi getting nutrients? Also, are these fungi grown in gas rather than medium?

3. Authors simulate microgravity and anticipate using these devices in space programs. How can media use be justified as it is replenished every 10 hours? Is there any change in approach when this device is used in the space mission?

4. Can the growth of fungi be quantified for table 1 and 2. This can add more value to the paper.

Minor comment:

Figure 7 needs caption.

What microscope was used to take the brightfield images?

Author Response

Responses to Reviewers’ comments:

1. Figure 9 which shows fungal growth in the gas channel is not clear. Authors can provide an outline in the big image to make the inset more clear.

Ad. 1. We thank the Reviewer for the comment. The Fig. 9 was improved – other lab-on-chip image was used (outlines of the microchannel are more clear now) and additional captions in the image area were introduced to directly show the channels interface.

2. Comsol simulation shows no flow in the gas chamber where the fungi grow. So how are the fungi getting nutrients? Also, are these fungi grown in gas rather than medium?

Ad. 2. That is right, no medium flow was in the gas chamber – only air gas diffusion through the via holes. Culturing medium that was delivered through the medium channel was a sterile distilled water, which was slightly entering the connecting channels to maintain appropriate humidity within the fungi growth area (gas channel exclusively).

The information about culturing medium (sterile distilled water) that was used in our experiments was added to the section “2.2. Object of the study – F. culmorum” and “2.5. Culturing system”.

3. Authors simulate microgravity and anticipate using these devices in space programs. How can media use be justified as it is replenished every 10 hours? Is there any change in approach when this device is used in the space mission?

Ad. 3. As mentioned in the manuscript (lines 148-153, 169-177), a powdered form of fungi was prepared as a part of our experimentation. According to the text provided in the subscript:

“When dealing desiccated material, cultivation research in LOCs can be initialized even with a 3-month delay in relation to nanosatellite integration. Based on this, dosing of the life-supporting media can be performed after the launch to the LEO, when the proper position, inclination and communication with the nanosatellite is achieved.”

no changes have to be done in our culturing system to assure future nanosatellite mission. Thanks to the powdered fungi form, a start of the medium flow and culture replenishing (e.g. every 10 hours) can be done after the rocket launch, when the lab-on-chip platform is ready to be operated by the astronaut on the ISS, or remotely, utilizing Dedicated-On-Board-Computer (DOBC) and radio commands during the bio-nanosatellite space mission.

4. Can the growth of fungi be quantified for table 1 and 2. This can add more value to the paper.

Ad. 4. The thank the Reviewer for that comment. The added the quantitative information about the fungi growth in the manuscript (now it is Table 2 and Table 3).

The growth of F. culmorum was assessed as percentage of microscopic image coverage. On the top of photo a grid was superimposed, resembling of Thom’s counting chamber. Coverage percentage was calculated as a sum of all the grid’s elements where the mycelium was observed.

Minor comment:

Figure 7 needs caption.

Thank you for that comment. The manuscript was checked carefully in the context of typos, section numbering and other mistakes.

What microscope was used to take the brightfield images?

An optical microscope (Leica DM750) with a Leica ICC50 W Microscope camera was used to take fungi images.    
The information about the microscope model was added to the manuscript.

 

Round 2

Reviewer 1 Report

The suggested corrections are appropriately implemented.