Multi-volume hemacytometer

Cell counting has become an essential method for monitoring the viability and proliferation of cells. A hemacytometer is the standard device used to measure cell numbers in most laboratories which are typically automated to increase throughput. The principle of both manual and automated hemacytometers is to calculate cell numbers with a fixed volume within a set measurement range (105 ~ 106 cells/ml). If the cell concentration of the unknown sample is outside the range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration. We have developed a new hemacytometer that has a multi-volume chamber with 4 different depths containing different volumes (0.1, 0.2, 0.4, 0.8 µl respectively). A multi-volume hemacytometer can measure cell concentration with a maximum of 106 cells/ml to a minimum of 5 × 103 cells/ml. Compared to a typical hemacytometer with a fixed volume of 0.1 µl, the minimum measurable cell concentration of 5 × 103 cells/ml on the multi-volume hemacytometer is twenty times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope value of cell number in multi-chambers) showed a wide measurement range (5 × 103 ~ 1 × 106 cells/ml) while reducing total cell counting numbers by 62.5% compared to a large volume (0.8 µl-chamber) hemacytometer.

Introduction 1 maximum measurable cell concentration to decrease or increase. A typical 0.1 µl fixed volume (most 1 used volume in hemacytometers) contains 10 ~ 500 cells at 10 5 ~ 5×10 6 cells/ml while a 0.2 µl fixed 2 volume contains 10 ~ 500 cells at 5×10 4 ~ 2.5×10 6 cells/ml. Therefore, if a sample of unknown 3 concentration is out of range of the hemacytometer, the sample must be prepared again by increasing 4 or decreasing the cell concentration. 5 To solve this problem, the current study proposed the use of a multi-volume hemacytometer. 6 This device counts cells in four chambers at volumes of 0.1, 0.2, 0.4, 0.8 µl as shown in Figure 1. A 7 multi-volume cell counter can measure cell concentrations ranging from 10 6 cells/ml to 5×10 3 cells/ml. 8 Compared to a hemacytometer having a fixed volume of 0.1 µl, the minimum measurable cell 9 concentration is 5×10 3 cells/ml which is twenty times lower. Additionally, the Multi-Volume Cell 10 Counting model (cell concentration calculation with the slope value of the cell number in multi-11 chambers) was also validated to measured cell concentration and increases the measurement speed as a 12 sample of only 1.5 µl can measure the wide range of cell concentrations. 13 14

Results 15
To compare the accuracy and range of cell concentration measurement between the 16 conventional hemacytometer and the proposed multi-volume hemacytometer, we measured five 17 samples at 10 3 , 5×10 3 , 10 4 , 10 5 , and 10 6 cells/ml. In the case of the multi-volume hemacytometer, there 18 are two cell concentration calculation models necessary to determine the cell concentration.

Fixed-Volume Cell Counting (FVCC) model 1
A hemacytometer has a ruled surface below the cover glass of 0.1 mm which limits the volume 2 of liquid suspension to 0.1 µl per one of nine squares and restricts it to accurately measure the 3 concentration of cells in the range of 2×10 5~2 .5×10 6 cells/ml [9]. As shown in Table 1, in the current 4 experiment the hemacytometer shows a large standard deviation (more than 15%) in the samples lower 5 than 10 5 cells/ml. The proposed device has several chambers with different volumes such as 0.1, 0.2, 6 0.4, and 0.8 µl to contain the multi-volume of cells in suspension. Figure 2 shows that the number of 7 cells varied in conjunction with the volume. The number of cells increased with the volume of the 8 chamber. As shown in Table 1, chamber 1, 2, 3, 4, and the MVCC model were used to measure cell 9 concentration. In the current experiments, chamber 1 in area 1 mm² contained a volume of 0.1 µl equal 10 to the volume of the standard hemacytometer. In the low volume chamber, the number of cells was not 11 great enough to calculate the cell concentration. In contrast, as the volume of the chamber increased, 12 the cells also increased enough to allow the cell concentration to be calculated. Therefore, chamber 4 13 has a wide measurement range and was able to be measured at the lowest concentration at 5×10 3 14 cells/ml. However, in the maximum cell concentration of 10 6 cells/ml, it was difficult to count a high 15 number of cells in a large volume (0.8µl) and repeat the measurement five times. Therefore, the user 16 must select the appropriate chamber volume according to the sample. concentration is lower than 15%, while the FVCC model indicates a higher standard deviation. As 26 shown in Figure 4, measurement using the conventional hemacytometer shows that when the sample concentration was low, the relative standard deviation of measuring cell concentration increased 1 significantly. The measurement range of the conventional hemacytometer is 10 6 ~ 10 5 cells/ml, and 2 using a 0.8 µl chamber showed a wide measurement range of 10 6 ~ 5×10 3 cells/ml. However, in 10 6 3 cells/ml, the hemacytometer using a 0.8 µl chamber had too many cells to count manually 4 (approximately 4000). Figure 4 shows how the proposed MVCC model reduced the number of cell 5 counts needed to measure cell concentration without reducing the measurement range. The MVCC 6 model measured 5×10 3~ 10 6 cells/ml within a 10% standard deviation and reduced the counting cell 7 number by about 62.5%. The MVCC model reduced the counting burden in large volumes as a total 8 volume of only 1.5 µl (0.1+0.2+0.4+0.8 µl) was needed to measure the cell concentration at a wide 9 range of concentrations. 10 As well known, the hemacytometer is a basic device used to count cells in most laboratories. A 11 lot of times the sample of cells must be prepare by increasing or decreasing the cell concentration 12 because the unknown sample of cells is outside the range of the hemacytometer. Thus, our study 13 proposed a novel device to measure cell concentration using a multi-volume hemacytometer. This 14 device is designed to have a multi-chamber that can contain multi-volume such as 0.1, 0.2, 0.4, and 0.8 15 µl, expanding the contained volume for a hemacytometer. Compared to a standard hemacytometer with 16 a fixed volume of 0.1 µl, the minimum measurable cell concentration for the multi-chamber 17 hemacytometer is 5×103 cells/ml, which is twenty times lower. 18 19

Discussion 20
As well known, the hemacytometer is a basic device used to count cells in most laboratories. 21 Many times a cell sample needs to be re-prepared by either increasing or decreasing the cell 22 concentration because the unknown cell concentration is outside the range of the hemacytometer. Thus, 23 our study proposed a novel device to measure cell concentration using a multi-volume hemacytometer. 24 This device is designed to have a multi-chamber that can contain multi-volume such as 0.1, 0.2, 0.4, 25 and 0.8 μl, expanding the contained volume for a hemacytometer. Compared to a standard hemacytometer with a fixed volume of 0.1 μl, the minimum measurable cell concentration for the multi-1 chamber hemacytometer is 5×10 3 cells/ml, which is twenty times lower.

Materials and Methods 22
Multi-volume hemacytometer design 23 The multi-volume hemacytometer is designed to address the problem of loading cell samples 24 in unknown concentrations which can fall outside the range of a standard hemacytometer, requiring the 25 sample to be re-prepared. The proposed design is able to define multiple volumes as it consists of 4 26 chambers with different depths as follows: chamber 1 depth 0.1 mm, chamber 2 depth 0.2 mm, chamber 3 depth 0.4 mm, and chamber 4 depth 0.8 mm which are connected within the same sheet. Each chamber 1 has an area of 3 × 3 mm², between the chambers there is a slope to avoid the cells sticking to the 2 perimeter of the chamber which causes the cells to spread continuously. It also has an inlet and vent 3 area. The counter chamber is made using the PDMS production process after which the PDMS sheets 4 are bonded on a slide glass. (Figure 1

Additional information 29
The authors declare no competing interests.     Table 1 Comparison of cell concentration measurements between a standard hemacytometer and a 1 multi-volume hemacytometer.  : Cell concentration measurement quality control pass (%RSD <15%) 5 Table 2 Total cell counts from cell concentration measurements.    Comparison of the percent of relative standard deviation of cell number in each concentration between a standard hemacytometer and a multi-volume hemacytometer with four chambers and the MVCC model.