Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations

Round 1

Reviewer 1 Report

In this manuscript, the authors described a method for isolation of HDL and Exosome from small volumes of serum samples. The authors of this manuscript described a problem like a sample volume without providing related literature that examined using smaller sample volumes to comprehend the need to do this technique. Was there any previous work and what are the challenges that authors aimed to solve?

The reviewer could not find any problem with working with 200 microliters of biofluid. Therefore, this paper is not suitable for publication because it is not going to add to the current knowledge.

Minor comments:

1- Authors described the importance of two targets in the introduction; however, they did not mention the challenges of isolating HDL and exosomes from small volumes of serum samples. Please modify. Also, consider shortening the introduction after incorporating these challenges in the introduction.

2.2. HDL solution: what is solution A? A Reader needs to know what kind of chemical reaction/interaction is happening!

Line 173: which sample has HDL level of“42±1.00”? and how it is obtained? Please clarify.

Author Response

Reviewer 1

In this manuscript, the authors described a method for isolation of HDL and Exosome from small volumes of serum samples. The authors of this manuscript described a problem like a sample volume without providing related literature that examined using smaller sample volumes to comprehend the need to do this technique. Was there any previous work and what are the challenges that authors aimed to solve?

The reviewer could not find any problem with working with 200 microliters of biofluid. Therefore, this paper is not suitable for publication because it is not going to add to the current knowledge.

Comment 1:

Authors described the importance of two targets in the introduction; however, they did not mention the challenges of isolating HDL and exosomes from small volumes of serum samples. Please modify. Also, consider shortening the introduction after incorporating these challenges in the introduction.

 

Response:

Nowadays, the most widely used method for HDL and exosome isolation is the ultracentrifugation method [1–5]. This approach has several drawbacks, including a long duration of procedure, labor intensive, and high equipment cost that limit its application in research and clinical needs [1,4,6,7]. In addition, several publications showed that the isolation of HDL and exosomes requires a large sample volume [1,8,9]. Thus, the limited sample amount and the access to the ultracentrifuge device sometimes become a major problem in research and clinical needs. In this study, therefore, we demonstrated an ultracentrifugation-free method to isolate HDL and exosome from serum samples with a variety of small volumes as well as their characterizations.

We have added this explanation on page 2 line 69-78 to read “Nowadays, the most widely used method for HDL and exosome isolation is the ultracentrifugation method [24–28]. This approach has several drawbacks, including a long duration of the procedure, labor intensive, and high equipment cost that limit its application in research and clinical needs [24,27,29,30]. In addition, several publications showed that the isolation of HDL and exosomes requires a large sample volume [24,31,32]. Thus, the limited sample amount and the access to the ultracentrifuge device sometimes become a major problem in research and clinical needs. In this study, therefore, we demonstrated an ultracentrifugation-free method to isolate HDL and exosome from serum samples with a variety of small volumes as well as their characterizations.”. The reference numbers have been reordered according to their appearance. The new references were added (24-32) on page 15, line 480-499. The number of references hereafter were changed accordingly.

References:

1. 1. Michell, D.L.; Allen, R.M.; Landstreet, S.R.; Zhao, S.; Toth, C.L.; Sheng, Q.; Vickers, K.C. Isolation of High-Density Lipoproteins for Non-Coding Small RNA Quantification. J. Vis. Exp. 2016, 2016, doi:10.3791/54488.
2. 2. Henderson, C.M.; Vaisar, T.; Hoofnagle, A.N. Isolating and Quantifying Plasma HDL Proteins by Sequential Density Gradient Ultracentrifugation and Targeted Proteomics. Methods Mol. Biol. Clifton NJ 2016, 1410, 105–120, doi:10.1007/978-1-4939-3524-6_7.
3. 3. Gardiner, C.; Vizio, D.D.; Sahoo, S.; Théry, C.; Witwer, K.W.; Wauben, M.; Hill, A.F. Techniques Used for the Isolation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey. J. Extracell. Vesicles 2016, 5, 10.3402/jev.v5.32945, doi:10.3402/jev.v5.32945.
4. 4. Ludwig, N.; Whiteside, T.L.; Reichert, T.E. Challenges in Exosome Isolation and Analysis in Health and Disease. Int. J. Mol. Sci. 2019, 20, 4684, doi:10.3390/ijms20194684.
5. 5. Ayala‐Mar, S.; Donoso‐Quezada, J.; Gallo‐Villanueva, R.C.; Perez‐Gonzalez, V.H.; González‐Valdez, J. Recent Advances and Challenges in the Recovery and Purification of Cellular Exosomes. Electrophoresis 2019, 40, 3036–3049, doi:10.1002/elps.201800526.
6. 6. Konoshenko, M.Yu.; Lekchnov, E.A.; Vlassov, A.V.; Laktionov, P.P. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. BioMed Res. Int. 2018, 2018, 1–27, doi:10.1155/2018/8545347.
7. 7. Kurian, T.K.; Banik, S.; Gopal, D.; Chakrabarti, S.; Mazumder, N. Elucidating Methods for Isolation and Quantification of Exosomes: A Review. Mol. Biotechnol. 2021, 63, 249–266, doi:10.1007/s12033-021-00300-3.
8. 8. Baranyai, T.; Herczeg, K.; Onódi, Z.; Voszka, I.; Módos, K.; Marton, N.; Nagy, G.; Mäger, I.; Wood, M.J.; El Andaloussi, S.; et al. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. PLoS ONE 2015, 10, 1–13, doi:10.1371/journal.pone.0145686.
9. 9. Cumba Garcia, L.M.; Peterson, T.E.; Cepeda, M.A.; Johnson, A.J.; Parney, I.F. Isolation and Analysis of Plasma-Derived Exosomes in Patients With Glioma. Front. Oncol. 2019, 0, 651, doi:10.3389/FONC.2019.00651.

 

Comment 2:

HDL solution: what is solution A? A Reader needs to know what kind of chemical reaction/interaction is happening!

 

Response:

We have discussed with the representative of Cell Biolabs Inc. (HDL isolation kit company) about the components of precipitation solution A. Unfortunately, it is a proprietary of the company. Thus, they could not tell us about the formulation of precipitation solution A.

The principle of HDL purification kit is mainly based on the presence of dextran sulfate that selectively precipitate HDL. The kit eliminates the requirement for ultracentrifugation in the purification of HDL. By employing this commercial kit, a series of precipitation and low-speed centrifugation procedures are used to purify the HDL particles [1].

We have added the explanation regarding the principle of HDL purification kit on page 12, line 342-345 to read “The principle of the HDL purification kit is mainly based on the presence of dextran sulfate that selectively precipitates HDL. By employing this commercial kit, a series of precipitation and low-speed centrifugation procedures were used to purify the HDL particles [33].”. The reference number has been reordered according to their appearance.

Reference:

1. 1. HDL Purification Kit. Available online: https://www.cellbiolabs.com/hdl-purification-kit (accessed on 10 October 2021).

 

 

Comment 3:

Line 173: which sample has HDL level of“42±1.00”? and how it is obtained? Please clarify.

 

Response:

HDL level of 42±1.00 belonged to sera that we used as the sample sources for HDL isolation. We measured the HDL level of sera using Beckman Coulter DxC 800 Chemistry Analyzer (Beckman Coulter, USA) (page 3, line 119-120). We need data of HDL level in serum samples to calculate the percent recovery of isolated HDLs.

We have clarified this by making some sentences become clearer on page 3, line 118-119 (method section) to read “HDL level in serum samples and isolated HDLs was measured by Beckman Coulter DxC 800 Chemistry Analyzer (Beckman Coulter, USA).” and page 5, line 200-201 (result section) to read “HDL levels of serum samples, HDL200 and HDL500 were 42±1.00, 39.3±1.86, and 39±1.53 mg/dL, respectively (Figure 1A).”.

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript entitled "Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations" authors described a methodology able to isolate HDL and exosomes from small sample volumes of serum can be  used to isolate HDL and exosomes. 

Authors reported a very interesting methodology addressing an important issues in diagnostics. The paper is well described and methodology rigorously reported. 

I have no concerns about the quality of the paper.

Author Response

Reviewer 2

In the manuscript entitled "Alternative Method for HDL and Exosome Isolation with Small Serum Volumes and Their Characterizations" authors described a methodology able to isolate HDL and exosomes from small sample volumes of serum can be used to isolate HDL and exosomes. 

Authors reported a very interesting methodology addressing an important issue in diagnostics. The paper is well described and methodology rigorously reported. 

I have no concerns about the quality of the paper.

Response:

Thank you for your comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

This manuscript describes the HDL and exosomes as an important source of biomarkers and the lack of enough sample volume and difficulty in its isolation were addressed. This is an organized manuscript that would be recommended for acceptance. Suggestions to improve this manuscript are listed below:

 

1. In introduction a reference needed to be added for the statement “Body fluids are a significant primary source of bio-markers for non-invasive clinical diagnostic needs”
2. In Materials and methods section:
   1. The materials used should be listed completely as a separate sub-section for materials
   2. In section 2.2, precipitation solution A needs to be described to the reader
   3. In section 2.4 the time has to be mentioned for how long the SDS-PAGE was run at 150V.
3. In Results and discussion section:
   1. The molecular weight marker used needs to be pointed out in figure
   2. Are there any positive controls used for DLS and was the system suitability standard pass the results needs to be mentioned
4. Grammatical errors need to be checked throughout the manuscript
5. Please maintain the reference style format to be consistent for all the references.
   1. Some of the references does not have volume and page numbers (Ref 10, 22)
   2. Please maintain consistent reference style

Author Response

Reviewer 3

This manuscript describes the HDL and exosomes as an important source of biomarkers and the lack of enough sample volume and difficulty in its isolation were addressed. This is an organized manuscript that would be recommended for acceptance. Suggestions to improve this manuscript are listed below: 

 

Comment 1:

In introduction a reference needed to be added for the statement “Body fluids are a significant primary source of bio-markers for non-invasive clinical diagnostic needs”

Response:

We have added 2 references (page 1, line 39, reference 2 and 3).

 

Comment 2:

In Materials and methods section:

1. The materials used should be listed completely as a separate sub-section for materials

Response:

We have added a sub-section for materials (2.1. Materials) (page 2-3, line 80-92).

 

2. In section 2.2, precipitation solution A needs to be described to the reader

Response:

We have discussed with the representative of Cell Biolabs Inc. (HDL isolation kit company) about the components of the precipitation solution A. Unfortunately, it is a proprietary of the company. Thus, they could not tell us about the formulation of the precipitation solution A.

The principle of the HDL purification kit is mainly based on the presence of dextran sulfate that selectively precipitate HDL. The kit eliminates the requirement for ultracentrifugation in the purification of HDL. By employing this commercial kit, a series of precipitation and low-speed centrifugation procedures are used to purify the HDL particles [1].

We have added the explanation regarding the principle of HDL purification kit on page 12, line 342-345 to read “The principle of the HDL purification kit is mainly based on the presence of dextran sulfate that selectively precipitates HDL. By employing this commercial kit, a series of precipitation and low-speed centrifugation procedures were used to purify the HDL particles [33].” The reference number has been reordered according to their appearance.

Reference:

1. 1. HDL Purification Kit. Available online: https://www.cellbiolabs.com/hdl-purification-kit (accessed on 10 October 2021).

 

 

3. In section 2.4 the time has to be mentioned for how long the SDS-PAGE was run at 150V.

Response:

SDS-PAGE was run at 150 V for 2 hours. This additional information has been added to the method section (page 4, line 139-140).

 

Comment 3:

In Results and discussion section:

1. The molecular weight marker used needs to be pointed out in figure

Response:

We have revised figures 2 (page 6) and 6 (page 9) by adding the protein ladder figures (originally from the ladder making company). “The molecular weight marker used had a range from 10.5 to 175 kDa (Figure 2A).” was added to page 5, line 221-222 and “A protein ladder reference (Vivantis Technologies, Malaysia, cat no. PR0602) (A)”; “The protein ladder employed ranged from 10 to 240 kDa (Figure 6A).” and “A protein ladder reference (Vivantis Technologies, Malaysia, cat no. PR0623) (A).” on page 6, line 232-233, page 9, line 293-294 and 299, respectively.

 

2. Are there any positive controls used for DLS and was the system suitability standard pass the results needs to be mentioned

Response:

In this study we had no positive control for DLS. We compared the particle size of exosomes we isolated to another similar publication that worked with the same commercial kit that did not explore a smaller sample volume.

Prior to the sample analysis, phosphate buffer saline (PBS) was run as a negative control to make sure that we had no peak of particle size and to make sure that the commercial PBS used was free from any particle contamination.

This explanation has been added on page 13, line 382-384, to read “Prior to the sample analysis, phosphate buffer saline (PBS) was run as a negative control to make sure that we had no peak of particle size and to make sure that the commercial PBS used was free from any particle contamination.”.

 

Comment 4:

Grammatical errors need to be checked throughout the manuscript

Response:

Grammarly for Microsoft Office software v.6.8.621 has been used for checking grammatical errors. The corrections were on page 1, line 21 and the last line; page 2, line 76-78; page 4, line 182; page 5, line 196-197and line 214; page 13, line 340.   

 

Comment 5:

Please maintain the reference style format to be consistent for all the references.

1. Some of the references does not have volume and page numbers (Ref 10, 22)
2. Please maintain consistent reference style

Response:

We have fixed all references and checked the consistency carefully (page 15-17, line 440-538).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors in this manuscript have answered the question of this reviewer regarding the purpose of this work in the introduction. The use of a commercial kit for isolation of HDL. They mentioned that using this kit allowed them to avoid ultrafiltration in sample preparation.

There are two concerns regarding the quality/novelty of this work:

- This is commercially available and it was reported in the literature for the use of HDL isolation as a routine method.

- It is also worth mentioning that they did not cite any of the work which is also provided on the company's website like Holzer, M. et al. (2016). Refined purification strategy for reliable proteomic profiling of HDL2/3:
impact on proteomic complexity. Sci. Rep. 6:38533.

Overall this reviewer does not see a value in publishing this work since readers of this journal are interested in new isolation/separation methods.. 

Author Response

Reviewer’s comments:

Authors in this manuscript have answered the question of this reviewer regarding the purpose of this work in the introduction. The use of a commercial kit for isolation of HDL. They mentioned that using this kit allowed them to avoid ultrafiltration in sample preparation.

There are two concerns regarding the quality/novelty of this work:

- This is commercially available and it was reported in the literature for the use of HDL isolation as a routine method.

- It is also worth mentioning that they did not cite any of the work which is also provided on the company's website like Holzer, M. et al. (2016). Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity. Sci. Rep. 6:38533.

Overall this reviewer does not see a value in publishing this work since readers of this journal are interested in new isolation/separation methods.

Response:

Thank you for the reviewer's comments and suggestions. What we presented in the manuscript was an initial part of our project. In the process of our initial research on ATR-FTIR-based biomarker discovery in aging, we encountered a problem regarding the limitation of serum sample volume. We worked with left-over specimens from a previous study with very limited volumes, each sample only having a volume of about 800 µL - 1 mL. Furthermore, we wanted to isolate HDL and exosomes simultaneously. Unfortunately, the most common method to isolate the two targets is ultracentrifugation which requires a larger sample volume and has some drawbacks. It seemed impossible to use the ultracentrifugation to isolate the two targets as we had a limitation in the volume of samples. Finally, to overcome this issue, we tried the commercial HDL isolation kit and ExoQuick to isolate the two targets by modifying the common protocol provided by the companies. We reduced the sample volume and the reagent volume used followed by various characterization processes. What we have done could bring some benefits, including (1) the elimination of ultracentrifugation during the isolation process, (2) possible use of as small as 50 µL and 200 µL for exosome and HDL isolation, and (3) saving commercial kits used leading to cost efficiency in research and clinical settings.

 

“In this study, the isolation of HDL and exosomes was carried out using the HDL purification kit (Cell Biolabs, USA, cat no. STA, 607) and ExoQuick™ Exosome Precipitation Solution (System Biosciences, USA, cat no. EXOQ5A-1). These two commercial kits are commonly used to isolate these two targets. The selected HDL purification kit has been used by several researchers and they have successfully used it for HDL isolation. However, all of them followed the manufacturer’s instructions without doing any modifications to reduce the sample volume and kit used [1–3]. According to the manual of the HDL isolation kit, 10 mL of serum or plasma is needed for HDL isolation [4], and sometimes it is difficult to obtain a specimen with the aforementioned volume. Regarding exosome isolation, several studies using ExoQuick have succeeded in isolating exosomes from 250 µL of serum or plasma by following the kit manufacturer's protocol without exploring the possibility of using smaller sample volumes [5–7]. In this study, the potential of the HDL isolation kit and ExoQuick to isolate these two important targets with small serum volumes was explored. By using a small sample volume, the volume of chemical or reagent used will be smaller. Thus, the advantages of our alternative methods are (1) the elimination of an ultracentrifugation need during the isolation process, (2) possible use of as small as 50 µL and 200 µL for exosome and HDL isolation, and (3) saving commercial kits used leading to cost efficiency in research and clinical settings. In addition to conventional characterizations, in this study, we used ATR-FTIR spectroscopy combined with PCA and Quick Compare Analysis to ensure that HDL and exosomes isolated from different volumes had similar biocomponents. Moreover, this characterization approach could also be used to monitor biological components of other isolated biological fractions using various separation methods.”

We have added this explanation in the section of discussion (page 14, line 414-438). We have also added three citations related to previous works provided on the company's website (Holzer et al. 2016, Gilad et al. 2019, and Aggarwal et al. 2021) as the comparison to our study.

 

References:

1. Holzer, M.; Kern, S.; Birner-Grünberger, R.; Curcic, S.; Heinemann, A.; Marsche, G. Refined Purification Strategy for Reliable Proteomic Profiling of HDL2/3: Impact on Proteomic Complexity. Sci. Rep. 2016, 6, 38533, doi:10.1038/srep38533.
2. Gilad, D.; Atiya, S.; Mozes-Autmazgin, Z.; Ben-Shushan, R.S.; Ben-David, R.; Amram, E.; Tamir, S.; Chuyun, D.; Szuchman-Sapir, A. Paraoxonase 1 in Endothelial Cells Impairs Vasodilation Induced by Arachidonic Acid Lactone Metabolite. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2019, 1864, 386–393, doi:10.1016/j.bbalip.2018.12.008.
3. Aggarwal, G.; May-Zhang, L.S.; Yermalitsky, V.; Dikalov, S.; Voynov, M.A.; Amarnath, V.; Kon, V.; Linton, M.F.; Vickers, K.C.; Davies, S.S. Myeloperoxidase-Induced Modification of HDL by Isolevuglandins Inhibits Paraoxonase-1 Activity. J. Biol. Chem. 2021, 297, 101019, doi:10.1016/j.jbc.2021.101019.
4. HDL Purification Kit Available online: https://www.cellbiolabs.com/hdl-purification-kit (accessed on 10 October 2021).
5. Martins, T.S.; Catita, J.; Rosa, I.M.; Da Cruz e Silva, O.A.B.; Henriques, A.G. Exosome Isolation from Distinct Biofluids Using Precipitation and Column-Based Approaches. PLoS ONE 2018, 13, 1–16, doi:10.1371/journal.pone.0198820.
6. Tang, Y.-T.; Huang, Y.-Y.; Zheng, L.; Qin, S.-H.; Xu, X.-P.; An, T.-X.; Xu, Y.; Wu, Y.-S.; Hu, X.-M.; Ping, B.-H.; et al. Comparison of Isolation Methods of Exosomes and Exosomal RNA from Cell Culture Medium and Serum. Int. J. Mol. Med. 2017, 40, 834–844, doi:10.3892/ijmm.2017.3080.
7. Serrano-Pertierra, E.; Oliveira-Rodríguez, M.; Rivas, M.; Oliva, P.; Villafani, J.; Navarro, A.; Blanco-López, M.C.; Cernuda-Morollón, E. Characterization of Plasma-Derived Extracellular Vesicles Isolated by Different Methods: A Comparison Study. Bioeng. Basel Switz. 2019, 6, E8, doi:10.3390/bioengineering6010008.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

The authors of this manuscript answered the concerns of the reviewer and the paper is suitable for publication.