Evaluation of the Regenerative Potential of Platelet-Lysate and Platelet-Poor Plasma Derived from the Cord Blood Units in Corneal Wound Healing Applications: An In Vitro Comparative Study on Corneal Epithelial Cells

Round 1

Reviewer 1 Report

1.      This in vitro study provides novel information on the biochemical characteristics of cord blood platelet lysate (CB-PL) and cord blood platelet poor plasma (CB-PPP) and on their regenerative potential on Wistar rats corneal epithelial cells. The manuscript requires extensive scientific professional editing of the English style and correction of typing mistakes.

2.      Scientific comments.

a.      Page 3, line 5 from bottom: Is 865 g for 15 min correct? I am surprised that this g value (notably, g, not rpm) was applied to generate PRP. Usually, much smaller g values (200 – 250 g) are used for PRP production. I would expect that 865 g for 15 min would generate a product between PPP and PRP. Please clarify.

b.      Page 4, formula (1) for PC Volume (ml). Using this formula with a typical platelet count in PRP (e.g. 400 x 10³/ul), one would obtain: 400 x 0.01 = 4, and then, by adding 5, one would obtain 9 ml. I understand that this is the target volume for CB-PC. If so, I do not see the reason to report 10³/ul in the denominator. Please clarify.

c.       Page 4, last sentence in paragraph 2.4. I am confused by the 780 g value used to prepare ‘control plasma’. 780 g is quite close to 865 g (see point a. above). Why using a slightly different centrifugation for this ‘control’? Is this ‘control plasma’ PRP or PPP?

d.      Page 6, line 2. WJ-MSC or CEC? Please clarify.

e.      The authors should indicate some limitations of their study, including the use of non human, rat CEC, which limits results transfer to clinical applications in humans.

3.      Other comments.

a.      Page 6, line 6 in paragraph 2.8. I believe ‘sides’ should be ‘slides’.

b.      Page 7, paragraph 2.9, line 6 from bottom: please check ’… 10,000 all events…’.

c.       Please use consistently the Greek letter alpha (not a, not A) with MEM.

d.      Please use consistently the acronym FACT-Netcord.

e.      Page 9, line 4. Please add μm after 0.22.

f.       Table 3. mm/Hg should be mm Hg. Check also throughout text.

g.      Results section. This section contains numerous, unnecessary textual repetitions of results reported in tables 2 and 3. Only important, critical values should be reported in the text.

h.      Page 13, paragraph 3.5, line 8. Please check ‘afte96 days’. Days or hours? Line 5 from bottom: ‘proliferation rates’ or ‘migration speed’?

i.        Page 14, caption of figure 3. Line 2: A16-2A0 should be A16-A20. Line 4: ‘after 5 days’ or ‘after 4 days’?

j.        Page 16. Valentinni should be Valentini.

k.      Whole manuscript: city and country of device and reagent manufacturers should be reported only on first occurrence.

Author Response

Hellenic Cord Blood Bank,

Biomedical Research Foundation

Academy of Athens,

4 Soranou Ephessiou Street

115 27, Athens, Greece

Date: 14/9/2022

Dear Reviewer 1,

Initially, we would like to thank you for the comprehensive evaluation that you performed on our submitted manuscript. Below you will find our responses to your comments.

This in vitro study provides novel information on the biochemical characteristics of cord blood platelet lysate (CB-PL) and cord blood platelet poor plasma (CB-PPP) and on their regenerative potential on Wistar rats corneal epithelial cells.

The manuscript requires extensive scientific professional editing of the English style and correction of typing mistakes.

Author’s Response:

We thank the reviewer for his valuable opinion. We have checked the whole manuscript and we performed any corrections required regarding the scientific language of the manuscript. all grammar or phrase errors were corrected using the Grammarly software. If the manuscript will be accepted from the journal, additional changes in language of the manuscript will be performed by the language editing department of the journal.

2. Scientific comments. .Page 3, line 5 from bottom: Is 865 g for 15 min correct? I am surprised that this g value (notably, g, not rpm) was applied to generate PRP. Usually, much smaller g values (200 – 250 g) are used for PRP production. I would expect that 865 g for 15 min would generate a product between PPP and PRP. Please clarify.

Author’s Response:

Initially, we would like to thank the reviewer for this valuable question. The reason why we are using this high initial g value in our first centrifugation step is because we are performing the PRP production in CBUs with an average volume of 139 ml (Table 2). For this purpose and because we want to fractionate the whole CBU it is required in the first centrifugation step to apply this high g value. G values of 200-250 are considered enough when PRP or PC is performed from a peripheral blood sample with volume of 15- 30 ml. And actually this is reasonable, because the higher the initial volume is, this means that also high PLT number is also existed, so greater centrifugational force is required to achieve the same result.

In addition, we have previously validated our protocol regarding the production of PRP, PC and PL from whole CBUs and we have already published the specific criteria for the production of PRP from CBUs and also the validations that we have performed. Please you can check our previous publications. Investigating the production of platelet lysate obtained from low volume Cord Blood Units: Focus on growth factor content and regenerative potential (10.1016/j.transci.2022.103465) and Selection Criteria of Cord Blood Units for Platelet Gel Production: Proposed Directions from Hellenic Cord Blood Bank (Bioengineering (Basel). 2021 Apr 27;8(5):53. doi: 10.3390/bioengineering8050053.)

1. Page 4, formula (1) for PC Volume (ml). Using this formula with a typical platelet count in PRP (e.g. 400 x 10³/ul), one would obtain: 400 x 0.01 = 4, and then, by adding 5, one would obtain 9 ml. I understand that this is the target volume for CB-PC. If so, I do not see the reason to report 10³/ul in the denominator. Please clarify.

Author’s Response:

The formula that we have currently validated is the following:

[PRP PLTs] is meaning the concentration of PLTs which is expressed in y value 10³ / μl (e.g 400 x 10³ / μl). The reason why we are using the 10³ / μl is because after the multiplication of [PRP PLTs ] with 0.0.1 is to have a result still expressed in 10³ / μl. In your example, if the [PRP PLTs] is 400 x 10³ / μl x 0.01 = 4 x 10³ / μl. So, the dominator is used to have only a number (e.g. 4) which it will be added to 5 in order to have the final obtained volume.

2. Page 4, last sentence in paragraph 2.4. I am confused by the 780 g value used to prepare ‘control plasma’. 780 g is quite close to 865 g (see point a. above). Why using a slightly different centrifugation for this ‘control’? Is this ‘control plasma’ PRP or PPP?

Author’s Response:

Once again, we want to thank the reviewer for the valuable comments that performed. In our centrifugation the 780  g corresponds to 1500 rpm, which actually is the typical applied force or rpm for plasma or serum isolation from peripheral or cord blood. Actually these settings are very common to the most hematological laboratories for the isolation of blood products such as plasma or serum in order to be used in other tests such as screeting tests for infectious disease. Also in our laboratory we used these settings for the isolation of plasma or serum. For serum isolation also it is required the whole blood or cord blood to be initially placed to specific blood vacutainers allowing the blood clot formation, in order to retrieve finally the serum. This is a very good question from the reviewer. In our situation, we thought about to use plasma samples for our comparisons regarding the growth factors, instead of serum. We have not performing any measurements regarding the PLTs concentration or other parameters in the isolated plasm samples, so we took into granted this protocol and after the plasma isolation we used this sample for the performance of the ELISA for the determination of the growth factor levels. Reasonably, an amount of PLTs will also be included in the plasma, but we cannot assume safely what this PLT number is exactly. For this purpose, we think that in our experimental procedure, the plasma samples should be better control group, and we are using a well-known protocol to isolate. In our future experiments, we will perform also PLTs count in plasma samples in order to very sure for their number. However, in these set of experiments we think that to have the plasma samples as control samples for the growth factor comparison between PC and PPP is more than enough and the readers will clearly understand the differences between the different samples. I hope that our answer fulfils your question, but if any further clarification is required, we are more than willing to inform and to reply to your comments.

2. Page 6, line 2. WJ-MSC or CEC? Please clarify.

Author’s Response:

We feel sorry for this misunderstanding. Of course the text and the formula were referred to CECs and not to WJ-MSCs. We have performed the above correction in the manuscript. Please you can check the revised manuscript.

1. The authors should indicate some limitations of their study, including the use of non human, rat CEC, which limits results transfer to clinical applications in humans.

Based on the reviewer comment, we have added the following limitations, in the last paragraph of the discussion section of the manuscript, page 22.

“Furthermore, the limitations of this study are appropriate to be discussed. In this study, CECs derived from Wistar rats were used for the performance of the experimental conditions. Rat-derived CECs offer an available source of cells, in order to assess the proliferation, migration and wound healing potential when CB-PL and CB-PPP are applied. However, further experimental procedures using CECs of human origin should be performed in order to obtain safer conclusions regarding the clinical utility of the CB derivatives. Obtaining CECs of human origin is a demanding task, requiring the appropriate documentation to be prepared and informed consent from the patients, to be acquired, to obtain tissue biopsies for CECs isolation purposes. On the other hand, animal-derived CECs represent an easy-to-access source of cells, share common features with the hu-man-derived cells, thus a series of different experimental procedures can be applied. Once, all data have been gathered and a specific mechanism of action focused on the beneficial properties of CB-derivatives, has been proposed, then a greater validation study can be performed using CECs of human origin.”

3. Other comments.
4. Page 6, line 6 in paragraph 2.8. I believe ‘sides’ should be ‘slides’.

Author’s Response:

We performed the change of sides to slides. The reviewer was right. Please check the revised manuscript.

2. Page 7, paragraph 2.9, line 6 from bottom: please check ’… 10,000 all events…’.

Author’s Response:

The following revision was performed in the main manuscript.

“On average a number of 10,000 all events were acquired for each sample.”

However, we have standardized  our flow cytometric protocol to obtain 10.000 all events, in order then to analyze our result.

1. Please use consistently the Greek letter alpha (not a, not A) with MEM.

Αuthor’s Response:

The reviewer was right. We have check the whole manuscript, and we performed the appropriate corrections where required. Please check the revised version of the manuscript.

1. Please use consistently the acronym FACT-Netcord.

Author’s Response:

The right acronym is FACT-NetCord, so the appropriate changes have been performed to the whole manuscript. Please check the revised version of the manuscript.

4. Page 9, line 4. Please add μm after 0.22.

Author’s Response:

We have added the μm in page 9, line 4. Please check the revised version of the manuscript.

3. Table 3. mm/Hg should be mm Hg. Check also throughout text.

Author’s Response:

We have performed the revision in table 3 and where it else was required in the main text. Please check th revised version of the manuscript.

3. Results section. This section contains numerous, unnecessary textual repetitions of results reported in tables 2 and 3. Only important, critical values should be reported in the text.

Author’s Response:

Based on the reviewer’s comment, we delete any unnecessary textual repetitions and we kept only the important parameters. Please check the revised version of the manuscript.

3. Page 13, paragraph 3.5, line 8. Please check ‘afte96 days’. Days or hours? Line 5 from bottom: ‘proliferation rates’ or ‘migration speed’?

Author’s Response:

The reviewer was right to the performed comments. We have corrected the 96 days to 96 hours and also in page 13, paragraph 3.5 line 8 from the bottom, we corrected the proliferation rates to migration speed. Please check the revised version of the manuscript.

3. Page 14, caption of figure 3. Line 2: A16-2A0 should be A16-A20. Line 4: ‘after 5 days’ or ‘after 4 days’?

Author’s Response:

We have corrected in page 14, caption of figure 3, line 2: 2A0 to A20. Also, we have corrected both in the caption and the discussion section the “5 days” to “4 days”. Please check the revised version of the manuscript.

16. Page 16. Valentinni should be Valentini.

Author’s Response:

We have performed the correction in the name of the author from “ Valentinni” to Valentini. Please check the revised version of the manuscript.

1. Whole manuscript: city and country of device and reagent manufacturers should be reported only on first occurrence.

Author’s Response:

We thank the reviewer for this comment. However, we are following the author’s guidelines of the journal, and that’s why we keep all this information. If any changes are required regarding the reagents, these can be later edited by the editorial team of the journal, if the manuscript will be accepted.

Furthermore the whole manuscript has been checked for grammar or phrase errors and has been successfully revised where it was necessary. In addition, all grammar or phrase errors were corrected using the Grammarly software. If the manuscript will be accepted from the journal, additional changes in language of the manuscript will be performed by the language editing department of the journal.

With the above performed revisions, we hope that the quality of the manuscript has been improved significantly, and therefore proceed to the next step of the publication process.

Yours sincerely,

Panagiotis Mallis, MSc, PhD

Associate Scientist,

Hellenic Cord Blood Bank,

Biomedical Research Foundation Academy of Athens

4 Soranou Ephessiou Str, Athens, Greece, 115 27

tel: +302106597340

mob: +306971616467

Hellenic Cord Blood Bank,

Biomedical Research Foundation

Academy of Athens,

4 Soranou Ephessiou Street

115 27, Athens, Greece

Date: 14/9/2022

Dear Reviewer 1,

Initially, we would like to thank you for the comprehensive evaluation that you performed on our submitted manuscript. Below you will find our responses to your comments.

This in vitro study provides novel information on the biochemical characteristics of cord blood platelet lysate (CB-PL) and cord blood platelet poor plasma (CB-PPP) and on their regenerative potential on Wistar rats corneal epithelial cells.

The manuscript requires extensive scientific professional editing of the English style and correction of typing mistakes.

Author’s Response:

We thank the reviewer for his valuable opinion. We have checked the whole manuscript and we performed any corrections required regarding the scientific language of the manuscript. all grammar or phrase errors were corrected using the Grammarly software. If the manuscript will be accepted from the journal, additional changes in language of the manuscript will be performed by the language editing department of the journal.

2. Scientific comments. .Page 3, line 5 from bottom: Is 865 g for 15 min correct? I am surprised that this g value (notably, g, not rpm) was applied to generate PRP. Usually, much smaller g values (200 – 250 g) are used for PRP production. I would expect that 865 g for 15 min would generate a product between PPP and PRP. Please clarify.

Author’s Response:

Initially, we would like to thank the reviewer for this valuable question. The reason why we are using this high initial g value in our first centrifugation step is because we are performing the PRP production in CBUs with an average volume of 139 ml (Table 2). For this purpose and because we want to fractionate the whole CBU it is required in the first centrifugation step to apply this high g value. G values of 200-250 are considered enough when PRP or PC is performed from a peripheral blood sample with volume of 15- 30 ml. And actually this is reasonable, because the higher the initial volume is, this means that also high PLT number is also existed, so greater centrifugational force is required to achieve the same result.

In addition, we have previously validated our protocol regarding the production of PRP, PC and PL from whole CBUs and we have already published the specific criteria for the production of PRP from CBUs and also the validations that we have performed. Please you can check our previous publications. Investigating the production of platelet lysate obtained from low volume Cord Blood Units: Focus on growth factor content and regenerative potential (10.1016/j.transci.2022.103465) and Selection Criteria of Cord Blood Units for Platelet Gel Production: Proposed Directions from Hellenic Cord Blood Bank (Bioengineering (Basel). 2021 Apr 27;8(5):53. doi: 10.3390/bioengineering8050053.)

1. Page 4, formula (1) for PC Volume (ml). Using this formula with a typical platelet count in PRP (e.g. 400 x 10³/ul), one would obtain: 400 x 0.01 = 4, and then, by adding 5, one would obtain 9 ml. I understand that this is the target volume for CB-PC. If so, I do not see the reason to report 10³/ul in the denominator. Please clarify.

Author’s Response:

The formula that we have currently validated is the following:

[PRP PLTs] is meaning the concentration of PLTs which is expressed in y value 10³ / μl (e.g 400 x 10³ / μl). The reason why we are using the 10³ / μl is because after the multiplication of [PRP PLTs ] with 0.0.1 is to have a result still expressed in 10³ / μl. In your example, if the [PRP PLTs] is 400 x 10³ / μl x 0.01 = 4 x 10³ / μl. So, the dominator is used to have only a number (e.g. 4) which it will be added to 5 in order to have the final obtained volume.

2. Page 4, last sentence in paragraph 2.4. I am confused by the 780 g value used to prepare ‘control plasma’. 780 g is quite close to 865 g (see point a. above). Why using a slightly different centrifugation for this ‘control’? Is this ‘control plasma’ PRP or PPP?

Author’s Response:

Once again, we want to thank the reviewer for the valuable comments that performed. In our centrifugation the 780  g corresponds to 1500 rpm, which actually is the typical applied force or rpm for plasma or serum isolation from peripheral or cord blood. Actually these settings are very common to the most hematological laboratories for the isolation of blood products such as plasma or serum in order to be used in other tests such as screeting tests for infectious disease. Also in our laboratory we used these settings for the isolation of plasma or serum. For serum isolation also it is required the whole blood or cord blood to be initially placed to specific blood vacutainers allowing the blood clot formation, in order to retrieve finally the serum. This is a very good question from the reviewer. In our situation, we thought about to use plasma samples for our comparisons regarding the growth factors, instead of serum. We have not performing any measurements regarding the PLTs concentration or other parameters in the isolated plasm samples, so we took into granted this protocol and after the plasma isolation we used this sample for the performance of the ELISA for the determination of the growth factor levels. Reasonably, an amount of PLTs will also be included in the plasma, but we cannot assume safely what this PLT number is exactly. For this purpose, we think that in our experimental procedure, the plasma samples should be better control group, and we are using a well-known protocol to isolate. In our future experiments, we will perform also PLTs count in plasma samples in order to very sure for their number. However, in these set of experiments we think that to have the plasma samples as control samples for the growth factor comparison between PC and PPP is more than enough and the readers will clearly understand the differences between the different samples. I hope that our answer fulfils your question, but if any further clarification is required, we are more than willing to inform and to reply to your comments.

2. Page 6, line 2. WJ-MSC or CEC? Please clarify.

Author’s Response:

We feel sorry for this misunderstanding. Of course the text and the formula were referred to CECs and not to WJ-MSCs. We have performed the above correction in the manuscript. Please you can check the revised manuscript.

1. The authors should indicate some limitations of their study, including the use of non human, rat CEC, which limits results transfer to clinical applications in humans.

Based on the reviewer comment, we have added the following limitations, in the last paragraph of the discussion section of the manuscript, page 22.

“Furthermore, the limitations of this study are appropriate to be discussed. In this study, CECs derived from Wistar rats were used for the performance of the experimental conditions. Rat-derived CECs offer an available source of cells, in order to assess the proliferation, migration and wound healing potential when CB-PL and CB-PPP are applied. However, further experimental procedures using CECs of human origin should be performed in order to obtain safer conclusions regarding the clinical utility of the CB derivatives. Obtaining CECs of human origin is a demanding task, requiring the appropriate documentation to be prepared and informed consent from the patients, to be acquired, to obtain tissue biopsies for CECs isolation purposes. On the other hand, animal-derived CECs represent an easy-to-access source of cells, share common features with the hu-man-derived cells, thus a series of different experimental procedures can be applied. Once, all data have been gathered and a specific mechanism of action focused on the beneficial properties of CB-derivatives, has been proposed, then a greater validation study can be performed using CECs of human origin.”

3. Other comments.
4. Page 6, line 6 in paragraph 2.8. I believe ‘sides’ should be ‘slides’.

Author’s Response:

We performed the change of sides to slides. The reviewer was right. Please check the revised manuscript.

2. Page 7, paragraph 2.9, line 6 from bottom: please check ’… 10,000 all events…’.

Author’s Response:

The following revision was performed in the main manuscript.

“On average a number of 10,000 all events were acquired for each sample.”

However, we have standardized  our flow cytometric protocol to obtain 10.000 all events, in order then to analyze our result.

1. Please use consistently the Greek letter alpha (not a, not A) with MEM.

Αuthor’s Response:

The reviewer was right. We have check the whole manuscript, and we performed the appropriate corrections where required. Please check the revised version of the manuscript.

1. Please use consistently the acronym FACT-Netcord.

Author’s Response:

The right acronym is FACT-NetCord, so the appropriate changes have been performed to the whole manuscript. Please check the revised version of the manuscript.

4. Page 9, line 4. Please add μm after 0.22.

Author’s Response:

We have added the μm in page 9, line 4. Please check the revised version of the manuscript.

3. Table 3. mm/Hg should be mm Hg. Check also throughout text.

Author’s Response:

We have performed the revision in table 3 and where it else was required in the main text. Please check th revised version of the manuscript.

3. Results section. This section contains numerous, unnecessary textual repetitions of results reported in tables 2 and 3. Only important, critical values should be reported in the text.

Author’s Response:

Based on the reviewer’s comment, we delete any unnecessary textual repetitions and we kept only the important parameters. Please check the revised version of the manuscript.

3. Page 13, paragraph 3.5, line 8. Please check ‘afte96 days’. Days or hours? Line 5 from bottom: ‘proliferation rates’ or ‘migration speed’?

Author’s Response:

The reviewer was right to the performed comments. We have corrected the 96 days to 96 hours and also in page 13, paragraph 3.5 line 8 from the bottom, we corrected the proliferation rates to migration speed. Please check the revised version of the manuscript.

3. Page 14, caption of figure 3. Line 2: A16-2A0 should be A16-A20. Line 4: ‘after 5 days’ or ‘after 4 days’?

Author’s Response:

We have corrected in page 14, caption of figure 3, line 2: 2A0 to A20. Also, we have corrected both in the caption and the discussion section the “5 days” to “4 days”. Please check the revised version of the manuscript.

16. Page 16. Valentinni should be Valentini.

Author’s Response:

We have performed the correction in the name of the author from “ Valentinni” to Valentini. Please check the revised version of the manuscript.

1. Whole manuscript: city and country of device and reagent manufacturers should be reported only on first occurrence.

Author’s Response:

We thank the reviewer for this comment. However, we are following the author’s guidelines of the journal, and that’s why we keep all this information. If any changes are required regarding the reagents, these can be later edited by the editorial team of the journal, if the manuscript will be accepted.

Furthermore the whole manuscript has been checked for grammar or phrase errors and has been successfully revised where it was necessary. In addition, all grammar or phrase errors were corrected using the Grammarly software. If the manuscript will be accepted from the journal, additional changes in language of the manuscript will be performed by the language editing department of the journal.

With the above performed revisions, we hope that the quality of the manuscript has been improved significantly, and therefore proceed to the next step of the publication process.

Yours sincerely,

Panagiotis Mallis, MSc, PhD

Associate Scientist,

Hellenic Cord Blood Bank,

Biomedical Research Foundation Academy of Athens

4 Soranou Ephessiou Str, Athens, Greece, 115 27

tel: +302106597340

mob: +306971616467

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscripts submitted by Mallis et al about the possible use of Cord blood platelet lysate and cord blood platelet poor plasma in the corneal healing process is very interesting. The study is correctly designed and technically sound. The analyses have been performed with the highest technical standards and the data are robust enough to draw conclusions.

About the data on viability test, were Corneal epithelial cells expanded with culture medium contained different concentrations of cord blood platelet lysate and cord blood platelet poor plasma or only with 20%?

Author Response

Hellenic Cord Blood Bank,

Biomedical Research Foundation

Academy of Athens,

4 Soranou Ephessiou Street

115 27, Athens, Greece

Date: 14/9/2022

Dear Reviewer 2,

Initially, we would like to thank you for the comprehensive evaluation that you performed on our submitted manuscript. Below you will find our responses to your comments.

The manuscripts submitted by Mallis et al about the possible use of Cord blood platelet lysate and cord blood platelet poor plasma in the corneal healing process is very interesting. The study is correctly designed and technically sound. The analyses have been performed with the highest technical standards and the data are robust enough to draw conclusions.

About the data on viability test, were Corneal epithelial cells expanded with culture medium contained different concentrations of cord blood platelet lysate and cord blood platelet poor plasma or only with 20%?

Author’s Response

Initially, we would like to thank the reviewer for the valuable comments and the opinion towards to our submitted manuscript. For all experimental procedures performed in this study, we used the 20% of CB-PL or CB-PPP to validate their beneficial potential. Previously in our laboratory, we have used different concentrations and we have concluded that the 20% is the best concentration, for the CECs proliferation and migration.

Furthermore the whole manuscript has been checked for grammar or phrase errors and has been successfully revised where it was necessary. In addition, all grammar or phrase errors were corrected using the Grammarly software. If the manuscript will be accepted from the journal, additional changes in language of the manuscript will be performed by the language editing department of the journal.

With the above performed revisions, we hope that the quality of the manuscript has been improved significantly, and therefore proceed to the next step of the publication process.

Yours sincerely,

Panagiotis Mallis, MSc, PhD

Associate Scientist,

Hellenic Cord Blood Bank,

Biomedical Research Foundation Academy of Athens

4 Soranou Ephessiou Str, Athens, Greece, 115 27

tel: +302106597340

mob: +306971616467

Author Response File: Author Response.docx